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Role of CX3CL1/Fractalkine in Osteoclast Differentiation and Resorption Keiichi Koizumi, Yurika Saitoh, Takayuki Minami, Nobuhiro Takeno, Koichi Tsuneyama, Tatsuro Miyahara, This information is current as Takashi Nakayama, Hiroaki Sakurai, Yasuo Takano, Miyuki of September 26, 2021. Nishimura, Toshio Imai, Osamu Yoshie and Ikuo Saiki J Immunol 2009; 183:7825-7831; Prepublished online 18 November 2009;

doi: 10.4049/jimmunol.0803627 Downloaded from http://www.jimmunol.org/content/183/12/7825

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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Role of CX3CL1/Fractalkine in Osteoclast Differentiation and Bone Resorption1

Keiichi Koizumi,2* Yurika Saitoh,* Takayuki Minami,* Nobuhiro Takeno,* Koichi Tsuneyama,†‡ Tatsuro Miyahara,§ Takashi Nakayama,¶ Hiroaki Sakurai,*† Yasuo Takano,‡ Miyuki Nishimura,ʈ Toshio Imai,ʈ Osamu Yoshie,¶ and Ikuo Saiki*†

The recruitment of osteoclast precursors toward and subsequent cell-cell interactions are critical for osteoclast dif- ferentiation. Chemokines are known to regulate and adhesion. CX3CL1 (also called fractalkine) is a unique mem- brane-bound chemokine that has dual functions for cells expressing its receptor CX3CR1: a potent chemotactic factor in its soluble form and a type of efficient cell adhesion molecule in its membrane-bound form. In this paper, we demonstrate a novel role of CX3CL1 in -induced osteoclast differentiation. We found that osteoclast precursors selectively expressed CX3CR1, whereas CX3CL1 is expressed by osteoblasts. We confirmed that soluble CX3CL1 induced migration of cells Downloaded from containing osteoclast precursors, whereas immobilized CX3CL1 mediated firm adhesion of osteoclast precursors. Furthermore, a blocking mAb against CX3CL1 efficiently inhibited osteoclast differentiation in mouse bone marrow cells cocultured with osteo- blasts. Anti-CX3CL1 also significantly suppressed in neonatal mice by reducing the number of bone-resorbing mature osteoclasts. Collectively, CX3CL1 expressed by osteoblasts plays an important role in osteoclast differentiation, possibly through its dual functions as a chemotactic factor and adhesion molecule for osteoclast precursors expressing CX3CR1. The http://www.jimmunol.org/ CX3CL1-CX3CR1 axis may be a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid , , and cancer . The Journal of Immunology, 2009, 183: 7825–7831.

one is a highly dynamic structure undergoing constant and osteoclast-associate receptor, while osteoblasts express their remodeling through the balance between bone resorption respective (1, 2). In particular, RANK ligand (RANKL), a trans- B by osteoclasts and bone formation by osteoblasts at the membrane glycoprotein of the TNF-␣ superfamily expressed by specialized sites called bone multicellular units (1, 2). Osteoclasts osteoblasts, plays the major role in osteoclast differentiation via 3 are tartrate-resistant (TRAP) -positive multinu- RANK by activating the osteoclastogenic cascade of transcription

cleated cells that are generated from osteoclast precursor cells of factors NF-␬B, AP-1 (c-Fos), and NF-ATc1 (1, 2). Osteoblasts by guest on September 26, 2021 the / lineage through close cell-cell interac- also produce M-CSF that promote the survival and proliferation of tions with osteoblasts (1, 2). It is now known that osteoclast pre- osteoclast precursors via c-Fms (1, 2). Nowadays, it is possible to cursors express cell surface receptors such as c-Fms (the tyrosine generate osteoclast-like cells in vitro from purified by ␬ kinase receptor for M-CSF), receptor activator of NF- B (RANK), a mixture of soluble RANKL and M-CSF, thus greatly simplifying the analysis of osteoclast differentiation. Chemokines are a group of structurally related cytokines that † *Division of Pathogenic Biochemistry, Institute of Natural Medicine, The 21st Cen- regulate the migration and activation of leukocytes and other types tury COE program, ‡Department of Pathology (I), Faculty of Medicine, and §Depart- ment of Toxicology, Faculty of Pharmaceutical Sciences, University of Toyama, of cells expressing a group of seven transmembrane G protein- Toyama, Japan; ¶Department of Microbiology, Kinki University School of Medicine, coupled receptors (3). According to the arrangement of the amino- Osaka, Japan; and ʈKAN Research Institute Inc., Kobe, Japan terminal conserved cysteine residues, the chemokines are classified Received for publication October 29, 2008. Accepted for publication October 8, 2009. into four subfamilies: CXC, CC, C, and CX3C (3). Several che- 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 mokines have been shown to play an important role in osteoclas- with 18 U.S.C. Section 1734 solely to indicate this fact. togenesis. For example, parathyroid strongly stimulates 1 This study was supported by a Grant-in-Aid for Young Scientists (No. 15790089), osteoblasts to express CCL2, which in turn can potently recruit Grant-in-Aids for Cancer Research (Nos. 16022224 and 16023225), a Grant-in-Aid osteoclast precursors (4). CCL2 and CCL5 are also potently in- for the 21st Century COE Program from Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and by Solution Oriented Research for duced by RANKL during osteoclast differentiation and strongly Science of Japan Science and Technology Corporation and High-Tech Research Cen- promote the formation of TRAPϩ multinuclear cells by an auto- ter Project for Private Universities: matching fund subsidy from MEXT, 2002–2006. crine/paracrine manner (5). CCL3 is also induced by RANKL dur- K.K. designed and performed research, analyzed data, and wrote the paper; Y.S., T.Min., N.T., K.T., T.Miy., T.N., H.S., Y.T., and M.N. performed research and an- ing osteoclast differentiation (6) and a known osteoclastogenic fac- alyzed data; T.I., O.Y., and I.S. designed research and wrote the paper. tor in (7). 2 Address correspondence and reprint requests to Dr. Keiichi Koizumi, Division of CX3CL1 (also called fractalkine) is a membrane-bound chemo- Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, kine and the only known member of the CX3C subfamily (3). Toyama 930-0194, Japan. E-mail address: [email protected] CX3CL1 can be cleaved from its membrane proximal site by the 3 Abbreviations used in this paper: TRAP, tartrate-resistant acid phosphatase; ES/BS, eroded surface/bone surface; N.Oc/B.Pm, osteoclast number/bone perimeter; Oc.S/ family of a disintegrin and metalloprotease ADAM10 and BS, osteoclast surface/bone surface; PO, peroxidase; RANK, receptor activator of ADAM17 (8, 9). Importantly, CX3CL1 is a dual function mole- NF-␬B; RANKL, RANK ligand. cule: it potently attracts CX3CR1-expressing cells by its soluble Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 form and efficiently mediates firm adhesion CX3CR1-expressing

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803627 7826 ROLE OF CX3CL1 IN OSTEOCLAST DIFFERENTIATION cells by its membrane-bound form even without activation of in- hamster anti-mouse CX3CL1 mAb (clone 5H8-4) (15) and PE-conjugated tegrins (10, 11). Therefore, the CX3CL1-CX3CR1 axis may be mouse anti-hamster IgG (BD Pharmingen), and analyzed on a FACSCalibur particularly useful in a biological situation where both attraction (BD Biosciences). and subsequent cell-cell interactions are required. In this paper, we Immunohistochemistry demonstrate for the first time that CX3CL1 expressed by osteo- Human bone biopsy specimens fixed with 20% buffered formalin and em- blasts plays an important role in osteoclastogenesis via CX3CR1 bedded into paraffin after decalcification were obtained from the surgical that are expressed by osteoclast precursors. files of the Department of Pathology, Faculty of Medicine, University of Toyama (Toyama, Japan). After deparaffinization, tissue sections (5-␮m Materials and Methods thick) were heated in a target retrieval solution (Dako) for 15 min using a Mice microwave oven. Tissue sections were treated with 3% H2O2 in TBS for 10 min to inhibit endogenous peroxidase and with 5% BSA for 5 min to block Female ddy strain mice, pregnant if required, were purchased from SLC. nonspecific sites. Tissue sections were sequentially stained with rabbit Eight-week-old mice were used for the isolation of bone marrow cells and anti-human CX3CL1 (1/200; Torrey Pines Biolabs), rabbit anti-human osteoclast precursors. Neonatal mice were used for the isolation of primary CX3CR1 (1/300; Torrey Pines Biolabs), goat anti- K (1/50; Santa osteoblasts and the treatment with anti-CX3CL1 Ab. All mice were kept under Cruz), or control rabbit IgG and with peroxidase (PO)-conjugated goat specific pathogen-free and laminar air flow conditions in the Laboratory for anti-rabbit IgG polymer (EnVision-PO for rabbit primary Abs; Dako) or Animal Experiments, Institute of Natural Medicine, University of Toyama PO-conjugated rabbit anti-goat IgG polymer (Histofine for goat primary (Toyama, Japan). This study was conducted in accordance with the guideline Abs; Nichirei). Finally, tissue sections were treated with diaminobenzidine for the Care and Use of Laboratory Animals of University of Toyama. and counterstained with hematoxylin. To clarify the nature of CX3CL1- Cells and CX3CR1-positive cells, a double-immunostaining method was per- formed in human bone samples. A rabbit polyclonal Ab against CX3CL1 Downloaded from Primary osteoclast precursors were isolated as described previously (12). In (1/200) and Envision-PO, which, visualized by diaminobenzidine (brown brief, femora and tibiae from 8-wk-old mice were flushed with ␣-MEM (ICN color), was used for primary staining. CX3CR1 (1/300) and EnVision-AP Biomedicals) to release bone marrow cells. Cells were incubated in ␣-MEM (Dako), which was visualized by fast blue (blue color) (Vector Laborato- supplemented with 10% FBS for 3 h and nonadherent cells enriched with ries), were used for secondary staining. To avoid interference due to double osteoclast precursors were collected. Primary mouse osteoblasts were isolated recognition of the primary Ab applied in the step of secondary Ab appli- as described previously (13). In brief, calvariae from 2-day-old mice were cation, the specimens were soaked in boiling water for 10 min. We pre- treated with 0.1% (Wako Pure Chemical) and 0.2% dispase (Godo liminarily confirmed the negative staining after soaking in boiling water by Shusei). Osteoblasts were collected and cultured in ␣-MEM containing 10% applying PO-conjugated secondary Abs against rabbit IgG. To evaluate the http://www.jimmunol.org/ FBS. Mouse and a mouse osteoclast precursor cell line RAW proportion of double-positive cells, the numbers of brown, blue, and dual- 264.7 were cultured in ␣-MEM supplemented with 10% FBS. colored cells were counted within visual fields at ϫ400 magnification for each specimen. This study was approved by the institutional ethics com- RANKL-induced differentiation of osteoclasts in vitro mittee and written informed consent was obtained from each patient. RANKL-induced differentiation of osteoclasts in vitro was performed as assay described previously (12). In brief, primary osteoclast precursors (nonad- herent mouse bone marrow cells, splenocytes, and RAW 264.7) were sus- For the chemotaxis assay of mouse marrow cells, we used pended in ␣-MEM supplemented with 10% FBS and cultured in a 24-well EZ-TAXIScan MIC-1000 (Effector Cell Institute) (16). In brief, cells were culture plate at 1 ϫ 106 cells per well. After 48 h, the culture medium was suspended in RPMI 1640 (Sigma-Aldrich) containing 0.1% BSA and 20 replaced with fresh culture medium with or without 100 ng/ml mouse re- mM HEPES, pH 8.0, and applied to the holder assembly. Murine recom- by guest on September 26, 2021 combinant soluble RANKL (Wako Pure Chemical). After 4 days, cells binant chemokine domain CX3CL1 (0ϳ1000 nM; R&D Systems) was in- were dehydrated with ethanol-acetone (1:1) for 1 min, dried, and stained at jected into the other side of the chamber. Cell migration was recorded for room temperature with TRAP staining solution. TRAP-positive cells ap- 1 h at 37°C using a charge-coupled device camera and a coaxial episcopic peared dark red. We counted TRAP-positive multinucleated cells contain- illumination system. For the chemotaxis assay of RAW 264.7 cells, we ing three or more nuclei as osteoclasts. used Transwell plates (8.0-␮m pore size; Nuclepore) essentially as de- scribed previously (17). Briefly, the filters of the upper wells were coated Effects of CX3CL1 on differentiation and CX3CR1 expression of with fibronectin (Iwaki Glass) on their lower surface. RAW 264.7 cells osteoclast precursors suspended at 1 ϫ 106 cells/ml in RPMI 1640 (Sigma-Aldrich) containing 0.1% BSA and 20 mM HEPES, pH 8.0, and applied to the upper wells, Osteoclast precursors (nonadherent mouse bone marrow cells) were cul- while the lower wells contained medium with or without murine recom- tured in ␣-MEM supplemented with 10% FBS with or without 10 ng/ml binant CX3CL1 (R&D Systems). After4hat37°C, cells that had migrated murine recombinant chemokine domain CX3CL1 (10 nM; R&D Systems). to the lower surface were counted. After 2 days, cells were stained with TRAP and RT-PCR analysis for CX3CR1 of the cells was performed. Cell adhesion assay RT-PCR Cell adhesion to immobilized CX3CL1 was measured as described previ- This was performed as described previously (14). Briefly, total RNAs were ously (10). Briefly, each well of the 96-well plate was precoated with extracted from cultured cells using TRIzol reagent (Invitrogen). First- anti-secretory alkaline phosphatase Ab (10 ␮g/ml) in PBS at 4°C over- ␮ night. After washing with PBS, nonspecific binding sites were blocked strand cDNA was prepared from RNA template (1 g) using oligo(dT)18 primer and SuperScript II reverse transcriptase (Invitrogen). Reverse tran- with adhesion buffer (RPMI 1640 containing 1% BSA and 20 mM HEPES, scription was performed at 42°C for 50 min and then at 70°C for 15 min. pH 7.4). The CX3CL1-secretory alkaline phosphatase fusion protein was PCR amplification was performed by denaturation at 94°C for 30 s, an- added to the wells. Primary osteoclast precursors (see above) were added 5 nealing at the indicated temperatures for 1 min, and extension at 72°C for to the wells (1 ϫ 10 cells/well), and incubated for 30 min at 37°C. After 105 s, using template cDNA and TaKara Ex Taq HS PCR kit (Takara washing, adherent cells in each well were counted under the microscope. In Shuzo). PCR products were electrophoresed on 1.7% agarose gels and some experiments, plates were preincubated for1hatroom temperature stained with ethidium bromide. The sequences of the PCR primers are as with or without rat anti-mouse CX3CL1 mAb (10 ␮g/ml, clone 126315; follows: GAPDH sense, 5Ј-GGTGAAGGTCGGTGTGAACGGATTT-3Ј R&D Systems) before assay. and antisense, 5Ј-AATGCCAAAGTTGTCATGGATGACC-3Ј (478 bp); CX3CR1 sense, 5Ј-TCATCACCGTCATCAGCATCG-3Ј and antisense, Osteoclast differentiation by coculture Ј Ј Ј 5 -TTGAGGCAACAGTGGCTGAAC-3 (511 bp); TRAP sense, 5 -TC Osteoclast differentiation by coculture was performed as described previ- Ј Ј CCCTGGTATGTGCTGG-3 and antisense, 5 -GCATTTTGGGCTGCT ously (18). Briefly, primary osteoblasts (see above) were suspended in Ј Ј Ј GA-3 (220 bp); CCR1 sense, 5 -CTACAAGAGCCTGAAGCAGTG-3 ␣-MEM supplemented with 10% FBS and cultured in 96-well plates (1 ϫ Ј Ј and antisense, 5 -GGTACTTCCAGAACCGTTCAC-3 (371 bp). 104 cells/well) overnight. Bone marrow cells were added to osteoblasts at 4 Flow cytometric analysis 1.6 ϫ 10 cells/well, and the cells were cocultured for 6 days in ␣-MEM supplemented with 10% FBS and vitamin D3 (10 nM). In some experi- Mouse primary osteoblasts isolated from calvariae of neonatal mice (see ments, rat anti-mouse CX3CL1 mAb (20 ␮g/ml, clone 126315; R&D Sys- above) were fixed with 4% paraformaldehyde, sequentially stained with tems) or control rat IgG (Jackson Laboratory) was added to the medium. The Journal of Immunology 7827

TRAP-positive cells containing more than three nuclei were counted as osteoclasts (18). Treatment of neonatal mice with anti-CX3CL1 mAb Neonatal mice (2 days old, n ϭ 3) were injected i.p. with 500 ␮g hamster anti-mouse CX3CL1 mAb (clone 5H8-4) (15) or control hamster IgG (Jackson Laboratory) daily for 5 days. On day 8, mice were killed and the left femurs were fixed in 70% ethanol, decalcified, and embedded in glycol methacrylate. Serial 3-mm thick sections were made longitudinally in the distal region of femur and stained with toluidine blue and TRAP staining solution. Histomorphometric measurements were made at ϫ400 in a min- imum of four optical fields in the secondary spongiosa area from the growth plate-metaphyseal junction using a semiautomatic image analyzing system (Osteoplan II; Carl Zeiss) linked to a light microscope. eroded surface/bone surface (ES/BS), osteoclast number/bone perimeter (N.Oc/ B.Pm), and osteoclast surface/bone surface (Oc.S/BS) were obtained. The nomenclature and symbols are those recommended by the Nomenclature Committee of the American Society for Bone and Mineral Research (19). Statistical analysis The Student’s two-tailed t test was applied to determine the significance of differences between groups, and a value of p Ͻ 0.05 was considered Downloaded from significant. Results CX3CR1 were highly expressed in osteoclast precursors but not mature osteoclasts

The nonadherent fraction of bone marrow cells and splenocytes is http://www.jimmunol.org/ commonly used as the source of primary osteoclast precursors (20). When nonadherent primary bone marrow cells were cultured for 4 days with or without recombinant RANKL, we observed numerous TRAP-positive multinucleated cells (mature osteoclasts) in the cultures treated with RANKL (Fig. 1A). By RT-PCR, we found that CX3CR1 was strongly expressed in untreated cells (os- teoclast precursors) but not in those treated with RANKL (mature osteoclasts). We next investigated whether CX3CR1 expression of osteoclast precursors but not mature osteoclasts is unique to the by guest on September 26, 2021 nonadherent fraction of bone marrow cells. As shown in Fig. 1B, similar expression patterns were also observed in osteoclast pre- FIGURE 1. Osteoclast precursors but not mature osteoclasts express cursors and mature osteoclasts derived from mouse splenocytes CX3CR1. A, Osteoclast differentiation in vitro. Nonadherent cells isolated and mouse osteoclast precursor cell line RAW 264.7. from mouse bone marrow cells were cultured without (a) or with (b) re- As another chemokine receptor (Fig. 1A), CCR2 was also se- combinant RANKL (100 ng/ml) for 4 days and stained for TRAP. White lectively expressed in untreated cells, whereas CCR1 was strongly arrowheads indicate TRAP-positive osteoclasts. Ac and B, RT-PCR anal- up-regulated in mature osteoclasts as described previously (21). ysis. Total RNA was prepared from nonadherent cells derived from mouse Because suitable Abs against mouse CX3CR1 were not available, bone marrow cells (Ac), mouse and mouse osteoclast precursors we performed immunohistochemical staining of CX3CR1 in hu- cell line RAW 264.7, and those treated with recombinant RANKL. RT- PCR was done for the indicated genes. GAPDH served as the loading man bone sections. As shown in Fig. 1C, we observed CX3CR1- control. C, Immunohistochemical staining of human bone tissue for positive cells in close colocalization with osteoblasts. These CX3CR1. a and b, Immature osteoclasts and the aggregation state of pre- CX3CR1-positive cells could be regarded as immature osteoclasts cursor osteoclasts (white arrowheads) with morphologic features such as and/or the aggregation state of osteoclast precursors from the mor- two or three nuclei, attachment to the bone, and the clear outlines of the cell phologic features such as two or three nuclei, attachment to the surface are strongly positive for CX3CR1. In contrast, the cells were al- bone, and a clear outline of the cell surface (Fig. 1Ca). Cathepsin most negative and/or slightly positive for . Black arrows, os- bone. c and d, Mature osteoclasts (arrowheads) existing in the ,ء ;K is a representative marker of mature osteoclasts and its expres- teoblasts sion gradually increases from immature to mature osteoclasts. bone are negative for CX3CR1 but not strong positive for cathepsin ϫ These CX3CR1-positive cells were almost negative and/or slightly K. Original magnification ( 200). Representative results from experiments positive for cathepsin K (Fig. 1Cb). In contrast, cells existing in the repeated at least three times are shown. OC, Osteoclast; pOC, osteoclast precursor; mOC, mature osteoclast. bone matrix and showing morphologic features of mature oste- oclasts, such as multinucleated giant cells with an unclear and undulating cell surface, were found to be negative for CX3CR1 expression but not strong positive for cathepsin K (Fig. 1C, c and first examined chemotactic responses to soluble CX3CL1 using d). These results suggested that CX3CR1 was highly expressed in TAXIScan, a real-time chemotaxis system (16). As shown in Fig. osteoclast precursors. 2A, soluble CX3CL1 vigorously induced migration of cells in the nonadherent fraction of mouse bone marrow cells containing os- CX3CL1 directly mediates the migration and adhesion but not teoclast precursors with a typical bell-shaped dose-response curve. differentiation of osteoclast precursors Separately, we confirmed vigorous migration of cells of RAW The possible expression of CX3CR1 by osteoclast precursors 264.7, a mouse osteoclast precursor cell line, to soluble CX3CL1, prompted us to examine their biological responses to CX3CL1. We using a conventional chemotaxis chamber assay (data not shown). 7828 ROLE OF CX3CL1 IN OSTEOCLAST DIFFERENTIATION

FIGURE 3. No direct effect of CX3CL1 stimulation on differentiation and CX3CR1 expression of osteoclast precursors in vitro. A, Osteoclast precursors derived from mouse bone marrow cells were cultured in the presence of recombinant RANKL (100 ng/ml; a) or recombinant CX3CL1

(10 nM; b) for 2 days and stained for TRAP. Asterisks indicate TRAP- Downloaded from positive osteoclasts. B, RT-PCR of osteoclast precursors cultured in the presence of recombinant CX3CL1 (10 nM) for 2 days was performed for CX3CR1genes. GAPDH served as the loading control.

CX3CR1-positive monocytes/macrophage-like cells, which are thought to be osteoclast precursors, was observed more often by dou- http://www.jimmunol.org/ ble staining. CX3CL1-positive osteoblasts were elongated, and their CX3CL1-positive osteoclast precursors closely contacted with the os- teoblasts on the bone surface (Fig. 4C). FIGURE 2. CX3CL1 induces chemotaxis and cell adhesion of osteoclast precursors. Because of the lack of suitable surface markers, we used the non- adherent fraction of fresh mouse bone marrow cells as the source of osteoclast precursors. A, Chemotaxis assay. Using a real-time chemotaxis assay appara- tus TAXIScan, chemotactic responses to the gradients generated by mouse recombinant chemokine domain CX3CL1 (0 ϳ 1000 nM) were recorded ev- by guest on September 26, 2021 ery 1 min. After 1 h, cells that had migrated toward the opposite side were counted and the chemotaxis index was calculated. B, Cell adhesion assay. Cells were added to wells precoated with CX3CL1. After 1 h, bound cells were counted. Rat anti-CX3CL1 mAb (clone 126315) and control rat IgG (Cont. IgG) were used at 10 ␮g/ml to ascertain the binding specificity. Data represents p Ͻ 0.05. Representative results from experiments repeated ,ء .the mean Ϯ SD at least three times are shown.

We next examined cell adhesion to immobilized CX3CL1. Oste- oclast precursors (Fig. 2B) as well as RAW 264.7 cells (data not shown) efficiently adhered to immobilized CX3CL1. Furthermore, a rat anti-mouse CX3CL1 mAb, but not control rat IgG, effectively suppressed the adhesion of cells to immobilized CX3CL1. These results supported that CX3CR1-expressing osteoclast precursors were highly responsive to CX3CL1. In contrast, soluble CX3CL1 did not directly the induce differentiation of osteoclast precursors (Fig. 3A), and also did not down-regulate its CX3CR1 expression in contrast with RANKL by RT-PCR (Figs. 1Ac and 3B). FIGURE 4. Close contacts with CX3CL1-positive osteoblasts and CX3CR1-positive osteoclast precursors in vivo. A, FACS analysis. Primary Close contact between osteoblasts and osteoclast precursors via osteoblasts isolated from neonatal mouse calvariae were stained for surface CX3CL1-CX3CR1 interaction on bone surface CX3CL1. FACS profiles by control hamster IgG (shaded area) and ham- The possible selective expression of CX3CR1 by osteoclast precur- ster anti-CX3CL1 mAb (clone 5H8–4; blue line) are shown. B, Immuno- sors prompted us to examine whether osteoblasts expressed its ligand histochemical analysis of human bone tissue. Osteoblasts colocalized with the bone are clearly positive for CX3CL1. Arrowheads indicate osteoblasts CX3CL1. We isolated osteoblasts from dissected neonatal mouse cal- and asterisks indicate bone. Original magnification, ϫ200. Representative variae and stained for CX3CL1. As shown in Fig. 4A, osteoblasts results from experiments repeated at least three times are shown. C, Close were indeed strongly positive for CX3CL1. Immunohistochemistry of contact of CX3CL1-positive osteoblasts (white arrowheads) and CX3CR1- human bone sections also confirmed CX3CL1 expression by the typ- positive osteoclast precursors (black arrows) by double staining for ical consecutive osteoblast regions on the bone surface (Fig. 4B). Fur- CX3CL1 (brown) and CX3CR1 (blue). Asterisks indicate bone. Original thermore, the juxtaposition of CX3CL1-positive osteoblasts and magnification, ϫ1000. The Journal of Immunology 7829 Downloaded from

FIGURE 6. Anti-CX3CL1 mAb inhibits bone resorption in vivo. Neo- http://www.jimmunol.org/ natal mice (n ϭ 3) were injected daily with control hamster IgG or hamster anti-CX3CL1 mAb (clone 5H8–4) for 5 days. After sacrifice, left femur sections were stained for TRAP and by toluidine blue. Three mice were analyzed for each group. A and B represent those treated with control IgG; C and D represent those treated with anti-CX3CL1 mAb. E, ; OZ, osteogenic zone; GP, growth plate; AC, articular . TRAP-positive elongated cells on the bone surface are activated mature osteoclasts, which mediate bone resorption and invade and penetrate bone bits at the site of ϫ ϫ

osteogenic zone. Original magnification: 4(A, C); 40 (B, D). Repre- by guest on September 26, 2021 sentative results from experiments repeated at least three times are shown.

duced by anti-CX3CL1 (Fig. 5C). These results are consistent with FIGURE 5. Anti-CX3CL1 mAb inhibits osteoclast differentiation in the notion that CX3CL1 is involved in the osteoblast-induced dif- vitro. Osteoclast differentiation was induced in mouse bone marrow cells ferentiation of osteoclasts. by coculture with primary osteoblasts in the presence of vitamin D3 for 6 days. Rat anti-CX3CL1 mAb (clone 126315) or control rat IgG was added Inhibition of bone resorption in vivo by anti-CX3CL1 mAb to the culture medium at 20 ␮g/ml. A, Picture of cocultured cells after To examine the role of CX3CL1 in osteoclast differentiation in TRAP-staining. Coculture was done in the presence of control rat IgG (a) vivo, we injected neonatal mice with the hamster anti-CX3CL1 b or rat anti-CX3CL1 mAb (clone 126315; ). Asterisks indicate TRAP- mAb or control hamster IgG daily for 5 days and examined TRAP- positive osteoclasts. B, Effect of anti-CX3CL1 mAb on the number of positive osteoclasts in the osteogenic zone of left femur. We ob- TRAP-positive osteoclasts. TRAP-positive multinuclear cells containing three or more nuclei were counted as mature osteoclasts. C, Effect of anti- served a slight decreases in the number of TRAP-positive cells CX3CL1 mAb on the surface area of TRAP-positive osteoclasts. Surface (N.Oc/B.Pm) in mice treated with anti-CX3CL1 mAb compared areas of TRAP-positive osteoclasts were analyzed by image analysis soft- with those treated with control IgG but the differences were not p Ͻ 0.05. Repre- statistically significant (Fig. 6, A and C, and Table I). This could be ,ء .ware (NIH Image). Data represents the mean Ϯ SD sentative results from experiments repeated at least three times are shown. due to numerous TRAP-positive cells in vivo such as immature osteoclasts and some monocytes. We therefore focused on TRAP- positive invading and penetrating cells in the bone pit that could be Inhibition of osteoclast differentiation in vitro by anti-CX3CL1 mAb The possible expression of CX3CR1 by osteoclast precursors and Table I. Inhibitory effects of anti-CX3CL1 mAb on bone resorption in mice the corresponding expression of its ligand CX3CL1 by osteoblasts prompted us to examine the role of CX3CL1 in osteoclast differ- N.Oc/B.Pm (/100 mm) ES/BS (%) Oc.S/BS (%) entiation. Mouse bone marrow nonadherent cells and primary os- Ϯ Ϯ Ϯ teoblasts were cocultured in the presence of vitamin D3. After 6 Control IgG 505.6 66.0 10.5 1.2 10.3 0.4 days, TRAP-positive multinuclear osteoclasts were counted (Fig. CX3CL1 Ab 440.5 Ϯ 36.2 6.4 Ϯ 1.1* 5.4 Ϯ 1.5* 5A). The rat anti-CX3CL1 mAb but not control rat IgG strongly Five hundred micrograms of anti-mouse CX3CL1 mAb or control IgG was in- suppressed the induction of TRAP-positive multinuclear oste- jected into the peritoneal cavity of three mice for 5 days. The bone resorption index was evaluated according to the Nomenclature Committee of the American Society for p Ͻ 0.05 vs ,ء .oclasts (Fig. 5B). The surface areas of TRAP-positive osteoclasts, Bone and Mineral Research’s rules. Data represent the mean Ϯ SD another indicator of osteoclast maturation, were also markedly re- control IgG. Data are representative of three independent experiments. 7830 ROLE OF CX3CL1 IN OSTEOCLAST DIFFERENTIATION regarded as activated mature osteoclasts in the process of bone Previously, several chemokines have been shown to promote resorption. We observed dramatic decreases in the percentage of osteoclast differentiation (5, 7, 26, 27). In particular, CCL2 is pro- bone surface covered by osteoclasts (Oc.S/BS) as well as that of duced by osteoblasts upon stimulation with eroded surface (ES/BS) in mice treated with anti-CX3CL1 mAb and attract osteoclast precursors via CCR2 (4). The expression of (Fig. 6, B and D, and Table I). These results clearly demonstrate CCR2 by osteoclast precursors is further up-regulated by stimula- that anti-CX3CL1 is capable of inhibiting the formation of mature tion with RANKL (6). Furthermore, CCL2 and CCL5 can induce osteoclasts in vivo. the formation of TRAP-positive multinuclear cells even in the ab- sence of RANKL, although these cells are negative for bone re- sorption (5). Yang et al. reported that paracrine interaction of Discussion CCL9 and CCR1 in osteoclasts had a genuine physiologic role in In the present study, we have presented strong evidence that os- osteoclast differentiation and that anti-CCL9 Ab inhibited oste- teoclast precursors but not mature osteoclasts express CX3CR1 oclast differentiation in rats (27). However, attention to the signif- (Fig. 1), whereas osteoblasts express its ligand CX3CL1 in both icant species differences in osteoclastogenic chemokines is needed mice and humans. In addition, close contact with these cells on the to develop a new therapeutic approach by inhibition chemokines bone surface was revealed (Fig. 4). The results suggest that and their receptor interaction in bone diseases because of a lack of CX3CL1, which is a dual functional chemokine, on osteoblasts CCL9 in human. works as an adhesion molecule toward osteoclast precursors. Fur- From the findings of the present study, osteoblasts of both mice thermore, blocking of the CX3CL1-CX3CR1 axis by anti- and humans, which constitutively express CX3CL1 at high levels CX3CL1 mAb strongly inhibited osteoblast-induced differentia- (Fig. 4), may also use the soluble form of CX3CL1 to attract os- Downloaded from tion of osteoclasts in vitro (Fig. 5) and the numbers of mature teoclast precursors and the membrane-bound CX3CL1 to adhere osteoclasts actively resorbing the bone in vivo (Fig. 6). In partic- with osteoclast precursors (Fig. 2). However, we observed no ular, the typical bone resorption parameters, Oc.S/BS (active os- clear-cut reduction in the gross numbers of TRAP-positive cells in teoclasts which dissolve bone) and ES/BS (pits on bone surface the bone of neonatal mice treated with anti-CX3CL1 (Fig. 6 and formed by active osteoclasts), were markedly decreased by the Table I). In contrast, mature bone-resorbing osteoclasts were treatment with anti-CX3CL1 (Table I). These results clearly dem- clearly decreased by anti-CX3CL1. Even though the role of http://www.jimmunol.org/ onstrate for the first time that the CX3CL1-CX3CR1 axis plays an CX3CL1 as an chemoattractant for osteoclast precursors is not important role in the osteoclast differentiation and bone resorption. formally excluded, these results suggest that the initial attraction of Unfortunately, we were unable to test the effect of anti-CX3CR1 to osteoclast precursors by osteoblasts is less dependent on CX3CL1, directly prove the involvement of CX3CR1 in the osteoclast dif- and the CX3CL1-CX3CR1 axis is more involved in the later stages ferentiation because of the lack of suitable neutralizing anti-mouse of osteoclast differentiation, i.e., after the initial interactions be- CX3CR1 Abs. Moreover, it would be interesting to study the bone tween osteoblasts with osteoclast precursors. Given that the mem- tissues of CX3CR1- or CX3CL1-knockout mice, even though no brane-bound form of CX3CL1 can induce firm adhesion of cells gross bone abnormalities have been reported in these mice to date.

expressing CX3CR1 even without intracellular signaling, the by guest on September 26, 2021 CX3CR1 is known to be expressed by NK cells, a subset of CX3CL1-CX3CR1 axis may be primarily involved in the stabili- monocytes/, and mature mucosal dendritic cells. zation of adhesion between osteoblasts and osteoclast precursors Therefore, it may be not so surprising that osteoclast precursors, that allows the close cell-cell interactions necessary for osteoclast which represent a cell lineage closely related to monocytes/mac- differentiation. Another possibility may be that CX3CL1 is re- rophages (22), do express CX3CR1. However, it is rather striking quired for the survival of osteoclast precursors (28). Obviously, that mature osteoclasts cease to express this receptor (Fig. 1). further studies are necessary to elucidate the exact role of the Therefore, the expression of CX3CR1 in the osteoclast lineage is highly regulated, suggesting its critical role at the stage of oste- CX3CL1-CX3CR1 axis in the osteoclast differentiation and bone oclast precursors and immature osteoclasts. In contrast, CX3CL1 resorption. is known to be expressed by activated endothelial cells (23), ma- Pathologic bone resorption is a serious problem in diseases such ture dendritic cells (24), and intestinal epithelial cells (25). As as (29), osteoporosis (30), and cancer bone shown in Fig. 4, CX3CR1-expressing monocytes/macrophage-like metastases (31). Previously, one of the present authors demon- cells closely colocalize with CX3CL1-positive osteoclasts on the strated that anti-CX3CL1 mAb significantly lowers the clinical bone surface. Collectively, given that CX3CL1 is a dual function arthritis score and reduces synovial inflammatory cell infiltration molecule, serving as a potent chemoattractant by its soluble form in the mouse model of collagen-induced arthritis (15). Our present and an efficient adhesion molecule by its membrane-bound form study has further shown that the treatment with anti-CX3CL1 Ab (Fig. 2), the CX3CL1-CX3CR1 axis may be particularly suited for can reduce the bone resorption by mature, activated osteoclasts. the initial attraction and the subsequent adhesion of osteoclast pre- Therefore, the blocking of the CX3CL1-CX3CR1 axis may be cursors by osteoblasts. useful for the therapy of rheumatoid arthritis in two ways: sup- However, the possibility that CX3CL1-positive osteoblasts di- pression of cellular infiltrates in inflamed synovium (32) and re- rectly induce osteoclast maturation and CX3CR1 down-regulation duction of bone resorption by mature osteoclasts in affected joints in vivo is unclear in the present study, although CX3CL1 stimu- (33). Similarly, the blocking of the CX3CL1-CX3CR1 axis may be lation alone did not affect the differentiation and CX3CR1 mRNA an attractive therapeutic strategy for other bone resorbing diseases expression of osteoclast precursors in vitro (Fig. 3). Therefore, such as osteoporosis (30) and cancer bone metastases (34), where further studies are needed to clarify the CX3CR1 expression and reduction of mature osteoclasts would be beneficial. Further stud- the osteoclast differentiation grade in the proximity of CX3CL1- ies are necessary to test these possibilities. producing cells using bone sections by multiple immunohisto- chemical staining of both CX3CR1 and cell differentiation mole- cules in osteoclast precursors and CX3CL1 in osteoblasts, Acknowledgments especially with the difficulty of multiple staining against of the We thank Kureha Special Laboratory (Tokyo, Japan) and Effector Cell bone section. Institute (Tokyo, Japan) for helpful advice and technical support. The Journal of Immunology 7831

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