Inhibitory Immunoglobulin-Like Receptors LILRB and PIR-B Negatively Regulate Osteoclast Development

This information is current as Yu Mori, Sukenao Tsuji, Masanori Inui, Yuzuru Sakamoto, of September 28, 2021. Shota Endo, Yumi Ito, Shion Fujimura, Takako Koga, Akira Nakamura, Hiroshi Takayanagi, Eiji Itoi and Toshiyuki Takai J Immunol 2008; 181:4742-4751; ;

doi: 10.4049/jimmunol.181.7.4742 Downloaded from http://www.jimmunol.org/content/181/7/4742

References This article cites 46 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/181/7/4742.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Inhibitory Immunoglobulin-Like Receptors LILRB and PIR-B Negatively Regulate Osteoclast Development1

Yu Mori,*† Sukenao Tsuji,* Masanori Inui,* Yuzuru Sakamoto,* Shota Endo,* Yumi Ito,* Shion Fujimura,* Takako Koga,‡ Akira Nakamura,* Hiroshi Takayanagi,‡ Eiji Itoi,† and Toshiyuki Takai2*

Osteoclasts, multinucleated cells of myeloid-monocytic origin, are responsible for bone resorption, which is crucial for maintenance of bone homeostasis in concert with bone-forming osteoblasts of nonhematopoietic, mesenchymal origin. Re- ceptor activator of NF-␬B ligand (RANKL) and M-CSF, expressed on the surface of and secreted by osteoblasts, respectively, are essential factors that facilitate osteoclast formation. In contrast to the activation processes for osteoclast formation, inhibitory mechanisms for it are poorly understood. Herein we demonstrate that inhibitory Ig-like receptors recruiting Src homology 2 domain-containing tyrosine phosphatase 1 (SHP-1) are expressed on osteoclast precursor cells like other myeloid Downloaded from cells, and that they play a regulatory role in the development of osteoclasts. We detected cell-surface expression of paired Ig-like (PIR)-B and four isoforms of leukocyte Ig-like receptor (LILR)B on cultured osteoclast precursor cells of mouse and human origin, respectively, and showed that all of these ITIM-harboring inhibitory receptors constitutively recruit SHP-1 in the presence of RANKL and M-CSF, and that some of them can suppress osteoclast development in vitro. Fluorescence energy transfer analyses have suggested that the constitutive binding of either murine PIR-B or its human http://www.jimmunol.org/ ortholog LILRB1 to MHC class I molecules on the same cell surface comprises one of the mechanisms for developmental regulation. These results constitute the first evidence of the regulation of osteoclast formation by cell-surface, ITIM-har- boring Ig-like receptors. Modulation of these regulatory receptors may be a novel way to control various skeletal system disorders and inflammatory arthritis. The Journal of Immunology, 2008, 181: 4742–4751.

one is continuously remodeled through bone formation Osteoblasts and other bone marrow stromal cells express two by osteoblasts and bone resorption by osteoclasts, both of essential factors for osteoclast differentiation (osteoclastogen- B which innovate the bone structure and sustain its struc- esis), namely receptor activator of NF-␬B ligand (RANKL),3 a tural and functional integrity. Osteoclasts are multinucleated cells member of the TNF family, and M-CSF (1, 2). RANKL is con- by guest on September 28, 2021 derived from bone marrow hematopoietic precursor cells of my- sidered to be a crucial cytokine that drives the development of eloid-macrophage lineage, whereas osteoblasts are of nonhemato- monocyte-macrophage-lineage cells into osteoclasts (3), while poietic, mesenchymal origin. It is of particular importance to elu- M-CSF is indispensable for the proliferation and survival of cidate the molecular mechanisms for bone homeostasis mediated osteoclast precursor cells (4). RANKL stimulation of osteoclast by osteoclasts and osteoblasts for the development of novel ther- precursor cells leads to recruitment of TNFR-associated factor apeutic strategies for various skeletal system disorders, such as (TRAF)6 to RANK, and activates key nuclear factors c-Fos and excessive bone formation (osteopetrosis), bone reduction (osteo- NF-␬B, which then induce the expression of for oste- porosis), and inflammatory arthritic diseases, including rheumatoid oclastogenesis governed by a key transcription factor, NFATc1 arthritis. (5). Mice deficient in RANKL, RANK, TRAF6, NF-␬B, or c- Fos show impaired osteoclast differentiation and osteopetrosis (1, 2, 6). In addition to this pivotal molecular sequence involv- *Department of Experimental Immunology, Institute of Development, Aging, and Cancer, and Center of Excellence Program for Innovative Therapeutic Development ing a cytokine, its receptor, intracellular signal mediators, and Towards the Conquest of Signal Transduction Diseases, Tohoku University, Sendai, transcription factors, we (7, 8) and others (9, 10) recently iden- Japan, and the Core Research for Evolutional Science and Technology Program, tified a novel and indispensable signal cascade initiated by two Japan Science and Technology Agency, Saitama, Japan; †Department of Orthopedic Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan; and ‡De- membrane-associated, homodimeric adaptor molecules, namely partment of Cell Signaling, Graduate School, Tokyo Medical and Dental University FcR␥ and DAP12, both of which harbor an ITAM that, upon and Center of Excellence Program for Frontier Research on Molecular Destruction tyrosine phosphorylation initiated by RANKL, conveys Syk- and Reconstruction of Tooth and Bone, Tokyo, Japan mediated calcium signaling pivotal for the induction of Received for publication August 16, 2007. Accepted for publication July 28, 2008. NFATc1. Several cell-surface receptors in the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by the Core Research for Evolutional Science and 3 ␬ ␤ Abbreviations used in this paper: RANKL, receptor activator of NF- B ligand; 2m, Technology Program of the Japan Science and Technology Agency, a grant-in-aid ␤ ␥ 2-microglobulin; BMM, bone marrow monocyte/macrophage-lineage cells; FcR , from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, common ␥ subunit; FRET, fluorescence resonance energy transfer; LILR, and a grant from the 21st Century Center of Excellence Program for Innovative leukocyte Ig-like receptor; MNC, multinucleated cells; MOC, multinucleated mature Therapeutic Development Towards the Conquest of Signal Transduction Diseases. osteoclast; PIR, paired Ig-like receptor; POC, prefusion osteoclast; SHP-1, Src ho- 2 Address correspondence and reprint requests to Dr. Toshiyuki Takai, Depart- mology 2 domain-containing tyrosine phosphatase 1; SIRP, signal regulatory recep- ment of Experimental Immunology, Institute of Development, Aging and Cancer, tor; TRAP, tartrate-resistant acid phosphatase. Tohoku University, Seiryo 4-1, Sendai 980-8575, Japan. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 4743 have also been suggested to be associated with FcR␥ or DAP12 1077 (Sigma-Aldrich), according to the guidelines of the Ethical Commit- ϩ on osteoclast precursor cells (7, 11). tee of Tohoku University Graduate School of Medicine. CD14 monocytes were then purified by positive selection with MACS (Miltenyi Biotec). To On the other hand, osteoprotegerin (12), SHIP (13), and SH2 ϩ generate multinucleated mature osteoclasts (MOCs), CD14 monocytes domain-containing tyrosine phosphatase 1 (SHP-1) (14) are were cultured at 5 ϫ 105 cells/ml in ␣-MEM, supplemented with 10% FCS suppressive elements for osteoclastogenesis (6), because tar- and penicillin-streptomycin, for 7 days in the presence of 100 ng/ml geted disruption or naturally occurring loss-of-function muta- RANKL and 20 ng/ml M-CSF (PeproTech). Tartrate-resistant acid phos- ϩ tion of the genes for the secretory or one of the intra- phatase (TRAP) multinucleated cells (MNCs, Ͼ3 nuclei) were counted. Prefusion osteoclasts (POCs) were obtained at 72 h of culture in the same cellular phosphatases resulted in increased osteoclastogenesis medium as that for MOCs. Macrophages were obtained by 7 days of cul- leading to osteoporosis. Osteoprotegerin is a secreted TNF re- ture of CD14ϩ monocytes in the medium containing M-CSF but not ceptor-related protein that acts as a decoy receptor that blocks RANKL. RANKL binding to RANK on osteoclasts (15), while SHIP in C57BL/6 (B6) mice were purchased from Charles River Japan. The Ϫ/Ϫ osteoclasts inhibits PI3K, an inducer of cell survival, cytoskel- generation of PIR-B-deficient (Pirb ) mice was described previously (31). Mice were maintained and bred in the animal unit of the Institute of etal rearrangement, and motility, and thus regulates the activa- Development, Aging, and Cancer (Tohoku University, Sendai, Japan), ac- v tion of osteoclasts (6, 16). Additionally, viable motheaten (me / cording to guidelines for experimental animals defined by The Institute of mev) mice, in which tyrosine phosphatase activity of cytosolic Development, Aging, and Cancer Animal Studies Committee. The induc- enzyme SHP-1 is partially lost (residual activity, 10–20%) (14), tion of murine osteoclasts in vitro was performed as described previously (7). Briefly, bone marrow cells were cultured at 5 ϫ 105 cells/ml in show severe defects in hematopoiesis and exhibit bone reduc- ␣-MEM with 10% FCS containing 10 ng/ml M-CSF. After 2 days, adher- tion due to an increased number of osteoclasts and the enhanced ent cells were used as bone marrow monocyte/macrophage lineage cells bone resorptive activity (14). It is suggested that SHP-1 plays a (BMMs). The BMMs were further cultured for 3 days in the presence of Downloaded from ϩ role in signals downstream of RANKL (17) as well as in a 100 ng/ml RANKL and 10 ng/ml M-CSF to generate MOCs. TRAP Ͼ pathway involving LPS stimulation (18). These observations MNCs ( 3 nuclei) were counted. POCs were obtained by a 48-h culture in the same medium as that for MOCs. Murine osteoblasts were prepared as indicate the presence of intracellular regulatory processes in- described previously (7). volving SHP-1 that inhibit relatively early stages of osteoclast development (6). However, little is known about the molecular Antibodies network for this inhibition, compared with the wealth of evi- The mAbs used for immunophenotypic and functional assays were as fol- http://www.jimmunol.org/ dence of the cell-surface receptors and intracellular activation lows: purified and FITC-conjugated anti-LILRB1 (clone GHI/75; BD cascades facilitating osteoclastogenesis (7, 11). Pharmingen), PE-conjugated and purified anti-LILRB2 (clone 42D1, rat In the immune system, SHP-1 activation takes place down- IgG2a, Beckman Coulter), purified and FITC-labeled anti-LILRB3 (clone stream of various inhibitory receptors, such as human leukocyte 222821; R&D Systems), purified and PC-5 conjugated anti-LILRB4 (clone ZM3.8; Beckman Coulter), anti-PIR-A/B (6C1; a generous gift from Drs Ig-like receptor (LILR)B (19–21), signal regulatory receptor Hiromi Kubagawa and Max D. Cooper, University of Alabama at Birming- (SIRP)1␣ (22), murine paired Ig-like receptor (PIR)-B (23, 24), ham), anti-SIRP␣ (clone P84; BD Pharmingen), anti-HLA-A/B/C (clone ␤ ␤ and gp49B (25, 26). SHP-1 is recruited, via its SH2 domain, to G46-2.6; BD Pharmingen), anti-human 2-microglobulin ( 2m) (clone b b the phosphotyrosine residues of the ITIM present in the cyto- TU¨ 99; BD Pharmingen), anti-H2K /D (clone 28-8-6; BD Pharmingen); ␤ and anti-mouse 2m (clone S19.8; BD Pharmingen). The Abs used for by guest on September 28, 2021 plasmic portions of these cell-surface receptors, and then it is immunoblot analysis were as follows: biotinylated anti-ILT3 (anti- activated through tyrosine phosphorylation by Src family ki- LILRB4, R&D Systems), and anti-phosphotyrosine (4G10) mAbs and anti- nases (27), leading to the inhibition of calcium mobilization SHP-1 Abs (Upstate Biotechnology). (28). It is tempting to suppose that functional expression of these inhibitory immunoreceptors also occurs on osteoclasts, Microarray analysis because in the immune system these inhibitory receptors are Microarray analysis was performed on Affymetrix U133 often coexpressed with the activating counterparts that associ- Plus 2.0 arrays using mRNAs prepared from purified monocytes, cultured ate with the ITAM-harboring membrane adaptors mentioned macrophages, POCs, and MOCs as described above. above (19, 29, 30). Indeed, our recent findings regarding the Flow cytometric analysis functional expression of some activating Ig-like receptors on murine osteoclasts in vitro, such as PIR-A and SIRP␤1 (7), Cell-surface expression of LILRs or PIR-B was assessed by flow cytomet- suggest the presence of counter-regulating receptors on murine ric analysis (FACS LSR, BD Biosciences) using the specific mAbs de- scribed above. For the detection of LILRB3 expression, the secondary as well as human osteoclasts. reagent was PE-labeled rat anti-mouse Abs (BD Pharmingen). For the de- In this paper, we report the expression and regulatory roles of tection of PIR-B expression, anti-PIR-A/B (6C1) labeled with Alexa Fluor isoforms of human LILRB, namely LILRB1, B2, B3, and B4 (also 647 (Invitrogen) was used. known as ILT2/LIR-1/CD85j, ILT4/LIR-2/CD85d, ILT5/LIR-3/ CD85a, and ILT3/LIR-5/CD85k, respectively), and murine PIR-B Assessment of osteoclastogenesis after Ab stimulation in osteoclasts derived from human peripheral blood and mouse Human CD14ϩ monocytes were obtained as described above. For bone marrow, respectively. Our findings strongly suggest that the crosslinking experiment, the anti-LILRB1 (GHI/75), LILRB3 (222821), and LILRB4 (ZM3.8) mAbs and isotype control Abs were converted to LILRB- and PIR-B-associated constitutive signaling mediated by Ј Ј F(ab )2 using an ImmunoPure IgG1 Fab and F(ab )2 preparation SHP-1 regulates the differentiation of osteoclast precursors in con- (Pierce). Twenty-four-well flat-bottom plates were coated overnight at 4°C cert with RANKL and M-CSF. Fluorescence resonance energy Ј ␮ ϩ with F(ab )2 at the concentration of 20 g/ml in PBS. CD14 monocytes transfer (FRET) examination revealed that the constitutive cis-in- were plated at 5 ϫ 105 per well and then cultured with 100 ng/ml RANKL and 20 ng/ml M-CSF for 7 days. Osteoclastogenesis was assessed by teraction of LILRB1 with its ligands, MHC class I molecules, on ϩ the surface of the same osteoclast precursor cells contributes to counting TRAP multinucleated cells. developmental regulation. Murine bone histology Materials and Methods Microcomputed tomography was performed using ␮CT40 (Scanco Medi- Cell culture cal). Murine skeletal tissues were fixed in paraformaldehyde, decalcified in EDTA, dehydrated in ethanol, and then embedded in paraffin. Sections Human PBMCs were obtained individually by separating peripheral blood were stained with H&E or for TRAP activity using a TRAP staining kit from healthy volunteers by density gradient centrifugation with Histopaque (TaKaRa Biomedicals). 4744 REGULATION OF OSTEOCLASTOGENESIS BY LILRB AND PIR-B

FIGURE 1. Expression of LILRs on myeloid-lineage cells. A, Microarray analysis of LILRs and activating Ig-like receptor TREM2 and OSCAR expression on myeloid-lineage cells derived from a healthy donor was performed on Affymetrix Human Genome U133 Plus 2.0 arrays using mRNAs prepared from CD14ϩ peripheral blood-derived monocytes (Mono), cultured macrophages (M␾), POCs, and MOCs. For LILRB mRNA expression, see the text for details. For LILRA mRNA expression, LILRA3 and LILRA4 mRNA expression was not detected in any lineages of cells examined. In monocytes, LILRA1, A2, A5, and A6 mRNA was distinctive. In macrophages,

LILRA1 and LILRA5 expression was reduced compared Downloaded from with that in monocytes, while the expression of LILRA2 and LILRA6 was sustained. In POCs, the expression levels of LILRAs, LILRA5 in particular, were decreased when compared with those in monocytes. In MOCs, the expres- sion profiles of LILRA1–A6 were roughly the same as those in POCs. B, Flow cytometric analysis of cell-surface

expression on myeloid-lineage cells stained with mAbs http://www.jimmunol.org/ against LILRB1 (GHI/75), LILRB2 (42D1), LILRB3 (22281), and LILRB4 (ZM3.8). Representative data are shown from three separate experiments with similar re- sults, each of which employed the set of myeloid-lineage cells prepared individually from three donors. by guest on September 28, 2021

Pit formation assay PIR-A/B, and protein-G conjugated Sepharose beads. Immunoprecipitates were separated by SDS-PAGE using 15% gels, followed by transfer to For assessment of the bone resorption activity of bone marrow cell-derived Ϫ/Ϫ polyvinylidene difluoride (Millipore) membranes. Each membrane was osteoclasts, bone marrow cells from B6 and Pirb mice were cultured on probed with HRP-conjugated anti-phosphotyrosine mAb and anti-SHP-1 calcium phosphate matrices for 8 days in the presence of various concen- mAb. The probed membranes were stripped with strip buffer and then trations of RANKL and 20 ng/ml M-CSF (PeproTech). After induction of reprobed with anti-LILRB1, anti-LILRB2, anti-LILRB3, anti-LILRB4, and the osteoclasts, the area of bone resorption was determined with image anti-PIR-A/B. analysis software (Adobe Photoshop 7.0). Statistical analysis Confocal microscopy and FRET analysis Statistical significance was assessed by means of Student’s t test, with p Ͻ Confocal laser scanning microscopy of human PBMC-derived POCs was 0.05 being considered significant. performed with Alexa Fluor 546-conjugated anti-LILRB1, LILRB2, ␤ LILRB3, LILRB4, and 2m mAbs, and Alexa Fluor 647-conjugated anti- HLA-A/B/C mAb. Confocal laser scanning microscopy of murine bone Results marrow-derived POCs was performed with Alexa Fluor 546-conjugated Four isoforms of LILRB are expressed on human osteoclasts ␣ ␤ anti-PIR-A/B, SIRP , and 2m mAbs, and Alexa Fluor 647-conjugated in vitro H-2Kb/Db mAb. All of these mAbs were directly labeled with an Alexa Fluor 546 or 647 monoclonal labeling kit (Invitrogen). POCs were visu- In the immune system, the ITAM-mediated activation signal is alized by FluoView FV1000 confocal microscopy (Olympus), which was often coupled with counter-regulation via ITIM-harboring recep- controlled with FV-10ASW software. Alexa Fluor 546/647 FRET imaging tors that recruit SHP-1 (27, 32, 33). In the present study, we first was performed with excitation at 543 nm and detection of donor Alexa attempted to determine whether the expression of inhibitory iso- Fluor 546 and FRET emission, followed by 633 nm excitation and acceptor Alexa Fluor 647 detection. After image acquisition, FRET efficiencies were forms of human LILR, LILRBs, could be detected on POCs or calculated using FV-10ASW software. FRET efficiency (E) was obtained MOCs in vitro. To this end, we first examined the gross profiles as follows: E ϭ 1 Ϫ IDA/ID ϫ 100 where IDA indicates the fluorescence of mRNA expression of LILRB molecules in human peripheral blood D intensity of prebleach donor cells and I the fluorescence intensity of post- monocytes, in vitro-cultured macrophages, POCs, and MOCs by bleach donor cells. E of Ͼ0.05 was defined as the threshold level for substantial transfer efficiency. means of microarray analysis. As shown in Fig. 1A, the mRNA expression profiles of the individual LILRBs exhibited several Immunoblot analysis characteristics for each cell lineage. As a common observation, For immunoprecipitation analysis, cell lysates were sequentially incu- high mRNA expression of LILRB1–B4 was observed for every bated with anti-LILRB1, anti-LILRB2, anti-LILRB3, anti-LILRB4, anti- lineage. In peripheral blood monocytes, the mRNA expression of The Journal of Immunology 4745

FIGURE 2. LILRBs show constitu- tive tyrosine phosphorylation and asso- ciation with SHP-1 and down-regulate osteoclast differentiation. A, Cellular ly- sates of human monocytes (Mono), macrophages (M␾), POCs, and MOCs were subjected to immunoprecipitation (IP) with LILRB mAbs. The immuno- precipitates were separated by SDS- PAGE and immunoblotted (IB) with anti-phosphotyrosine (PY) (4G10), anti- SHP-1, and anti-LILR mAbs. Re- presentative data are shown from three separate experiments with similar re- sults, each of which employed the set of myeloid-lineage cells prepared individ- ually from three donors. B and C, Down-regulation of osteoclastogenesis by LILRB stimulation. Human CD14ϩ monocytes were plated on dishes coated Downloaded from Ј with F(ab )2 of LILRB1, LILRB3, LILRB4, or the isotype control murine IgG at the concentration of 20 ␮g/ml overnight at 4°C. After cell culture with RANKL and M-CSF for 7 days, TRAP staining was performed (B) and TRAPϩ

MNCs (n Ͼ 3) were counted (C). Rep- http://www.jimmunol.org/ resentative data are shown from three separate experiments with similar re- sults, each of which employed the set of myeloid-lineage cells prepared individ- ually from three donors.

LILRB1–B4 was distinctive. In macrophages prepared by cultur- ing osteoclastogenesis by means of immunoblot analysis (Fig. ing monocytes with M-CSF for 6 days, LILRB4 and LILRB5 ex- 2A). Cellular lysates of human monocytes, macrophages, POCs, pression was markedly enhanced, while LILRB1–B3 expression and MOCs were subjected to immunoprecipitation with LILRB by guest on September 28, 2021 was not remarkably altered. In POCs after 4 days of culture of Abs. Immune complexes were separated by SDS-PAGE and monocytes with RANKL and M-CSF, a positive control RANK then immunoblotted with either LILRB, phosphotyrosine, or mRNA was significantly induced, while the LILRB1–B4 mRNA SHP-1 Abs. In monocytes, the LILRB1, B3, and B4 level was relatively unchanged. In MOCs after 6 days of culture of were each represented by two molecular species, with the monocytes with RANKL and M-CSF, the expression profile of smaller ones being abundant. The apparent molecular masses of LILRB1–B5 was roughly the same as those in the case of POCs. the species were as follows: LILRB1, 95 and 90 kDa; LILRB3, Thus, through this microarray analysis, we verified the expression 95 and 85 kDa; and LILRB4, 60 and 53 kDa (not depicted in the of significant amounts of LILRB1–B4 mRNAs during the devel- figure). In contrast, macrophages, POCs, and MOCs predomi- opment of osteoclasts, suggesting the possible involvement of nantly had the larger protein species of each LILRB. The larger these inhibitory receptors in human osteoclastogenesis. Although LILRB species were identified as the tyrosine phosphorylated we monitored in parallel the profiles of mRNA expression of the forms of the proteins, as shown in phosphotyrosine blots (Fig. activating receptors, LILRAs, finding, for example, that the ex- 2A). Our attempts to identify LILRB2 protein in the immuno- pression of LILRA2 and LILRA6 was significant in both POCs precipitates with LILRB2 mAb (42D1) were not successful, and MOCs, we hereafter focus on the analysis on LILRBs in this possibly due to the intrinsic incapability of the mAb for immu- study. noblot. However, we consistently observed a phosphotyrosy- Next we attempted to detect the cell-surface expression of lated ϳ100-kDa protein species in the immunoprecipitates (Fig. LILRB1–B4 on immature osteoclasts, POCs, by flow cytometry 2A), indicative of LILRB2 phosphorylation in macrophages, (Fig. 1B). Fully matured MOCs in culture were not suitable for this POCs, and MOCs as for other LILRB isoforms. analysis because they were too large for the instrument. The ex- It is known that LILRB1 and LILRB4 recruit SHP-1 in mono- pression levels of LILRB1, LILRB2, and LILRB3 on POCs were cytes and dendritic cell subsets, as other inhibitory Ig-like re- comparable to those on monocytes and macrophages. It is inter- ceptors such as killer Ig-like receptors do (19, 34). We found esting that the LILRB4 levels on POCs were higher than those on that the LILRB1, B2, B3, and B4 immunoprecipitates contained monocytes and macrophages. Collectively, the flow cytometric 62-kDa SHP-1, as shown in each anti-SHP-1 blot (Fig. 2A). The analyses revealed that LILRB1, B2, B3, and B4 were distinctly amount of SHP-1 coimmunoprecipitated with either LILRB1, expressed on the surface of POCs, suggesting the inhibitory roles LILRB2, or LILRB4 was higher in macrophages, POCs, and of these receptors. MOCs than in monocytes. The amount of SHP-1 coprecipitated with LILRB3 was comparable among the lineages of cells de- Tyrosine phosphorylation of LILRB during osteoclastogenesis spite the abundant phosphotyrosylated species of LILRB3 in To further characterize the LILRB protein expression in oste- macrophages, POCs, and MOCs, suggesting that the SHP-1 as- oclasts, we examined their tyrosine phosphorylation profile dur- sociation with LILRB3 was, at least in part, independent on 4746 REGULATION OF OSTEOCLASTOGENESIS BY LILRB AND PIR-B Downloaded from

FIGURE 3. cis-Association between MHC class I molecules and LILRB1 or LILRB2 on POCs. A, Colocalization of either LILRB1 or LILRB2 and HLA- A/B/C, as seen on FRET analysis. From left to right, excited donor (Alexa Fluor 546-conjugated mAbs to LILRB1 (GHI/75), LILRB2 (42D1), LILRB3 (222821),

␤ ¨ http://www.jimmunol.org/ LILRB4 (ZM3.8), and 2m (TU99)) images (green), excited acceptor (Alexa Fluor 647-conjugated HLA-A/B/C mAb (G46-2.6]) images (red), FRET signal ␤ images (pseudocolor), and differential interference contrast (DIC) images are shown. FRET images indicate the cis-association between LILRB1 or 2m and HLA-A/B/C. Red to blue color indicators represent strong to weak FRET. B, Energy transfer between different pairs was detected as the increase in donor fluorescence after acceptor photobleaching. FRET efficiency (%) ϭ (1 Ϫ fluorescence intensity of prebleach donor cells/fluorescence intensity of postbleach donor cells) ϫ 100. FRET efficiency Ͼ5% was defined as the threshold level for substantial transfer efficiency. Data are the means Ϯ SEM for at least 25 POCs prepared .FRET effect considered to be significant ,ء .from one donor receptor phosphotyrosylation. We failed to detect any recruit- B3, and B4 in POCs and MOCs were constitutively tyrosine ment of SHP-2 or SHIP to these LILRB proteins in each lineage phosphorylated and were constitutively associated with

of cells (data not shown). Thus, we concluded that LILRB1, B2, SHP-1. by guest on September 28, 2021

FIGURE 4. PIR-B in osteoclasts shows constitutive tyrosine phosphorylation and association with SHP-1. A, Expression of PIR-B on osteoclasts but not on osteoblasts. Flow cytometric analysis of cell-surface PIR-A and PIR-B expression on macrophages (M␾), POCs, and cultured osteoblasts from B6 (black lines) and PirbϪ/Ϫ (gray lines) mice, stained with anti-PIR-A/B mAb (6C1). Gray lines indicate PIR-A expression. The mean fluorescence intensity for each peak for macrophages and POCs is shown in the panels. In osteoblasts, PIR-A/B expression was not detected. B and C, Constitutive tyrosine phosphorylation of, and SHP-1 association with, PIR-B in osteoclasts. Cellular lysates of murine macrophages (M␾), POCs, and MOCs were subjected to immunoprecipitation (IP) with anti-PIR-A/B mAb. The immunoprecipitates were separated by SDS-PAGE and immunoblotted (IB) with anti-phosphotyrosine (PY) mAb (4G10) followed by anti-PIR-A/B blotting (B) or anti-SHP-1 blot (C). PIR-B (ϳ130 kDa) is constitutively tyrosine phosphorylated and is recruiting SHP-1 in POCs and MOCs. The Journal of Immunology 4747

FIGURE 5. PIR-B-deficient oste- oclasts exhibit up-regulated differen- tiation and enhanced bone resorption in vitro. A, Bone marrow-derived monocyte/macrophage progenitors (BMMs) derived from B6 and PirbϪ/Ϫ mice were cultured in the presence of 0, 10, or 100 ng/ml RANKL and 20 ng/ml M-CSF for 7 days followed by TRAP staining. B, Number of TRAPϩ multinuclear cells developed from BMMs of B6 and PirbϪ/Ϫ mice. After BMM culture, TRAPϩ multinucleated (n Ͼ 3) cells Ϯ were counted. Data are the means Downloaded from SD of triplicate determinations and are representative of three indepen- dent experiments with similar results. C, BMMs derived from B6 and PirbϪ/Ϫ mice were cultured on cal- cium phosphate matrices in the pres- ence of 0, 10, or 100 ng/ml RANKL http://www.jimmunol.org/ and 20 ng/ml M-CSF. D, The percent- age resorption pit area was measured as described in Materials and Meth- ods. Data are the means Ϯ SD of triplicate determinations and are rep- resentative of three independent ex- periments with similar results. by guest on September 28, 2021

Down-modulation of osteoclast differentiation by LILRB LILRB1 and LILRB2, as well as their murine counterpart PIR-B, aggregation are known to recognize a broad range of classical and nonclassical Constitutive phosphotyrosylation of LILRB1, B2, B3, and B4, and MHC class I molecules (35, 36), while neither LILRB3 nor their association with SHP-1 during osteoclastogenesis, prompted LILRB4 binds MHC class I molecules (37). Recently, we verified us to examine whether osteoclast development in vitro might be that human LILRB2 can bind HLA-A/B/C on the surface, namely suppressed by forced stimulation of LILRB molecules on mono- cis-binding, of the same human basophilic leukemia cells, KU812, cytes. We coated culture dishes with F(abЈ) of each LILRB mAb on confocal microscopy and FRET (38). Likewise, we also dem- 2 b b and then plated CD14ϩ monocytes and cultured them in the pres- onstrated that murine PIR-B recognizes H-2K /D in cis on the ence of RANKL and M-CSF. As shown in Fig. 2, B and C, stim- same mast cell surface (38). These preceding observations led us ulation of LILRB1, LILRB3, and LILRB4 significantly inhibited to examine whether cis-interaction between LILRB1 or LILRB2 the development of MOCs compared with that with the isotype and MHC class I molecules could be demonstrated on osteoclast control mAbs. LILRB4 aggregation caused the most significant precursor cells. To obtain any evidence for cis-binding between inhibition of the formation of TRAPϩ MOCs (Fig. 2, B and C). LILRB1 or LILRB2 and MHC class I, we performed FRET anal- However, this LILRB stimulation did not cause any impairment of ysis with each Alexa 546-conjugated anti-LILRB mAb as an en- the cytoskeletal structure when examined by F-actin staining (data ergy donor and Alexa 647-conjugated anti-HLA-A/B/C mAb as an not shown). Thus, it was suggested that LILRB1, B3, and B4 could acceptor, since the efficiency of the FRET from donor to acceptor have the potential to inhibit osteoclastogenesis upon interaction represents a very sensitive indicator of the molecular colocaliza- with their cognate ligands in vitro. Thus far, our attempts to dem- tion on a scale of 2–10 nm (39). Fig. 3 summarizes the donor- onstrate a possible inhibitory role of LILRB2 in osteoclastogenesis acceptor FRET effect observed on the confocal laser microscopy. were not successful, possibly due mainly to a limited variety of When POCs were doubly stained with Alexa 546-conjugated LILRB2-specific mAbs (data not shown). LILRB mAb (green) and Alexa 647-conjugated HLA-A/B/C mAb (red), and then excited with a 532-nm laser, we detected strong cis-Binding between MHC class I molecules and LILRB1 and FRET from the excited LILRB1 and LILRB2 mAb signal to the LILRB2 on the POC cell surface HLA-A/B/C mAb signal (7.5 Ϯ 0.6 and 12.5 Ϯ 0.7%, respec- ␤ The possible LILRB-mediated regulation of osteoclastogenesis tively). As a positive control, 2m showed strong energy transfer suggests a crucial role of the interaction with their cognate ligands. with HLA-A/B/C (12.0 Ϯ 1.2%). In contrast, negative control 4748 REGULATION OF OSTEOCLASTOGENESIS BY LILRB AND PIR-B Downloaded from

FIGURE 6. cis-Binding between PIR-B and MHC class I molecules on POCs. A, Colocalization of PIR-B and H-2Kb/Db observed on FRET analysis

␣ ␤ http://www.jimmunol.org/ is shown. From left to right, excited donor images (Alexa Fluor 546-conjugated PIR-A/B mAb (6C1), SIRP mAb (P84), and 2m mAb (S19.8)) (green), excited acceptor images (Alexa Fluor 647-conjugated H-2Kb/Db (28-8-6)) (red), FRET signal images (pseudocolor), and differential interference contrast (DIC) images are shown. FcR␥-deficient mice (Fcer1gϪ/Ϫ) exhibit no cell-surface expression of PIR-A. FRET images indicate the cis-binding between Ϫ/Ϫ b b ␤ b b PIR-B (Fcer1g POC) and H-2K /D or between 2m and H-2K /D . B, Energy transfer for each pair was detected as the increase in donor fluorescence after acceptor photobleaching. FRET efficiency (%) ϭ (1 Ϫ fluorescence intensity of prebleach donor cells/fluorescence intensity of postbleach donor FRET effect considered ,ء .cells) ϫ 100. FRET efficiency of Ͼ5% was defined as substantial transfer efficiency. Data are the means Ϯ SD for at least 40 cells to be significant.

LILRB3 and LILRB4 did not show a significant energy transfer concentration of RANKL. The resorption pit area produced on by guest on September 28, 2021 with HLA-A/B/C on POCs (2.3 Ϯ 0.4% and 3.5 Ϯ 0.4%, re- calcium phosphate matrices was also larger for the culture of PIR- spectively). These findings strongly suggest that LILRB1 and B-deficient MOCs than for PIR-B-sufficient B6 mice (Fig. 5, C and LILRB2, but not LILRB3 or LILRB4, can associate with MHC D), indicating that PIR-B deletion may lead to accelerated bone class I molecules in cis on the POC surface, as was shown for resorption primarily due to increased onsteoclasts. These analyses LILRB2 binding to MHC class I molecules on a basophilic indicate that murine osteoclastogenesis in the presence of RANKL leukemia cell line (38). and M-CSF in vitro is also negatively regulated by PIR-B, as was the case for human LILRB1, B3, and B4 described above. Deletion of PIR-B leads to accelerated osteoclastogenesis in vitro cis-Association between MHC class I molecules and PIR-B on the murine POC cell surface To circumvent the lack of a suitable model system for verifying any physiological role of LILRB in bone remodeling, we utilized The finding of cis-binding of LILRB1 to MHC class I molecules -targeted mice lacking PIR-B (31), the murine counterpart of on the same POC cell surface shown in Fig. 3 led us to examine human LILRB1 and LILRB2 (36). First, we monitored the cell- whether the murine LILRB1/B2 counterpart PIR-B could also bind surface expression of PIR-B on murine POCs and osteoblasts in MHC class I molecules on the same POC surface in cis. Again, we culture by flow cytometry (Fig. 4A). As expected from the pre- performed FRET analysis with Alexa 546-conjugated anti-PIR- ceding microarray data (7), the PIR-B protein was abundantly ex- A/B mAb as an energy donor and Alexa 647-conjugated anti-H- pressed on POCs as well as on macrophages, but not on 2Kb/Db mAb as an acceptor. Fig. 6A shows that a FRET effect was osteoblasts. clearly detected for PIR-B and H-2Kb/Db as well as for PIR-A/B Next, samples of PIR-B-sufficient or -deficient macrophages, and H-2Kb/Db. A negative control, SIRP␣, a receptor for CD47, POCs, and MOCs were immunoblotted with Abs against PIR, did not show any significant FRET effect with H-2Kb/Db, while a ␤ b b phosphotyrosine, and SHP-1 (Fig. 4, B and C). As was the case for positive control, 2m, yielded distinct FRET with H-2K /D , con- human LILRBs (Fig. 2A), PIR-B was constitutively tyrosine phos- firming that the FRET analysis was valid. Fig. 6B summarizes the phorylated in POCs and MOCs, as was the case for murine B cells FRET effect for each donor-acceptor combination. These findings and other myeloid cells (36, 24, 40). We detected constitutive strongly suggest that PIR-B can bind to MHC class I molecules in SHP-1 association with PIR-B in POCs and MOCs (Fig. 4C). cis on the POC surface, as was the case for LILRB1 and LILRB2 We then cultured bone marrow cells from PIR-B-deficient or on POCs (Fig. 3) and for PIR-B on the mast cell surface (38). -sufficient mice in the presence of M-CSF and RANKL and com- pared the osteoclastogenesis. As shown in Fig. 5, A and B, MOC PIR-B-deficient mice are not osteoporotic development was significantly accelerated in PIR-B-deficient cells Finally, we compared bone histology and bone morphometric pa- in the presence of either a lower (10 ng/ml) or higher (100 ng/ml) rameters of the tibia of B6 and PirbϪ/Ϫ mice at various ages. We The Journal of Immunology 4749 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. PIR-B deficiency does not lead to osteoporosis. A, Histomorphologic analysis of sections of tibial metaphyses from 1.5-mo-old B6 and PirbϪ/Ϫ mice. Histologic sections of tibiae were stained for H&E. Original magnifications: upper, ϫ10; lower, ϫ50. Representative data are shown for three mice in each group with similar results are. B, Micro CT three-dimensional images of tibial proximal metaphyses of 3-mo-old B6 and PirbϪ/Ϫ mice. Representative images are shown from three mice in each group with similar results. PirbϪ/Ϫ mice did not exhibit an apparent reduction in bone mass. C, Quantitative analysis of trabecular bone expressed as percentages of trabecular bone volume vs total marrow space, osteoclast number and their surface area, and bone resorption rate in B6 and PirbϪ/Ϫ mice aged 1.5, 3, and 6 mo. D, Quantitative analysis of osteoblast surface area, bone formation rate, osteoid thickness, and mineral apposition rate in B6 and PirbϪ/Ϫ mice aged 1.5, 3, and 6 mo. Data are the means Ϯ SD (n ϭ 3 for mice of each age). No significant difference was observed between B6 and PirbϪ/Ϫ mice. anticipated a decreased bone mass, namely osteoporosis, in PIR-B blast function in terms of osteoblast surface area, bone formation deficiency, as was observed for SHP-1-mutant viable motheaten rate, osteoid thickness, or mineral apposition rate (Fig. 7D). These (mev/mev) mice (14); unexpectedly, however, we failed to demon- results indicate that the deletion of PIR-B does not significantly strate any osteoporosis on either bone histology at 1.5 mo of age alter either the osteoclast or osteoblast function in vivo, at least up (Fig. 7A) or micro CT analyses at 3 mo of age (Fig. 7B), a sig- to 6 mo of age. nificant decrease in bone volume, or an increase in osteoclast num- ber or their surface area, or in bone resorption rate in PirbϪ/Ϫ mice Discussion at any age examined (Fig. 7C). Osteoblastic parameters of the tibia LILRB1 and LILRB2 are human orthologs of murine PIR-B be- of PirbϪ/Ϫ mice also showed no significant alterations in osteo- cause of the similarity in their chromosomal localization, overall 4750 REGULATION OF OSTEOCLASTOGENESIS BY LILRB AND PIR-B structure, and ligand-binding specificities (36, 41). Murine ho- hand, LILRB4 polymorphisms have been screened for their pos- mologs or orthologs of human LILRB3 and LILRBB4 have not sible association with atopy, but no apparent association was de- been identified. In this study, we demonstrated that human tected (46). The association of either LILRB3 or LILRB4 with LILRB1, B2, B3, and B4 are expressed on cultured osteoclast pre- bone disorders, such as rheumatoid arthritis or bone metabolic dis- cursor cells derived from peripheral blood monocytes, and that eases, including osteopetrosis and osteoporosis, has not been re- LILRB1/B3/B4 could be inhibitory for osteoclast development in ported so far. Our present study immediately raises the question of the presence of RANKL and M-CSF. We also showed that one of whether any polymorphisms in LILRB1, B2, B3, or B4 are linked the inhibitory mechanisms could comprise the constitutive cis- with any bone disorders. Examination of these polymorphisms and binding of LILRB1 to MHC class I molecules on the same oste- their association with immune and osseous disorders will increase oclast precursor cells, leading to the constitutive recruitment of our understanding of the physiological roles of LILRBs. In par- SHP-1. A role of LILRB3 and LILRB4 was also shown to be ticular, LILRB4 could be a novel therapeutic target for inflamma- down-modulation of osteoclast development through their associ- tory bone diseases and osteoclastogenic disorders because of its ation with SHP-1, as in the case of LILRB1, but we could not relatively preferential expression on osteoclast precursor cells as analyze possible mechanisms further due to the lack of information well as its significant inhibitory role in differentiation into mature on their ligands. osteoclasts. In contrast to the distinct expression of LILRB1, B2, B3, and B4 on the osteoclast surface, expression of the activating isoforms, Acknowledgments LILRAs, was not prominent, at least in their mRNA levels. On We thank Nicholas Halewood for editorial assistance, and Akiko Sugahara- microarray analysis, the tendency was observed that the expression Tobinai for the outstanding technical support. Downloaded from of inhibiting LILRBs dominated that of activating LILRAs con- comitant with differentiation into osteoclasts as well as into mac- Disclosures rophages from monocytic cells. The dominant expression of The authors have no financial conflicts of interest. LILRBs over LILRAs may be required for proper regulation of osteoclast development. Additionally, these observations may in- References 1. Teitelbaum, S. L. 2000. Bone resorption by osteoclasts. Science 289: 1504–1508. dicate a putative role of activating LILRAs in the enhancement of http://www.jimmunol.org/ 2. Suda, T., N. Takahashi, N. Udagawa, E. Jimi, M. T. Gillespie, and T. J. Martin. osteoclast development, as demonstrated by the accelerated oste- 1999. Modulation of osteoclast differentiation and function by the new members oclastogenesis upon stimulation of murine receptors, including of the tumor necrosis factor receptor and ligand families. Endocr. 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