MIP-1γ Promotes Receptor Activator of NF-κ B Ligand-Induced Osteoclast Formation and Survival This information is current as Yoshimasa Okamatsu, David Kim, Ricardo Battaglino, of September 24, 2021. Hajime Sasaki, Ulrike Späte and Philip Stashenko J Immunol 2004; 173:2084-2090; ; doi: 10.4049/jimmunol.173.3.2084 http://www.jimmunol.org/content/173/3/2084 Downloaded from References This article cites 29 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/173/3/2084.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology MIP-1␥ Promotes Receptor Activator of NF-␬B Ligand-Induced Osteoclast Formation and Survival1 Yoshimasa Okamatsu,*† David Kim,* Ricardo Battaglino,* Hajime Sasaki,* Ulrike Spa¨te,* and Philip Stashenko2* Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes, and have also been reported to modulate osteoclast differentiation from hemopoietic precursor cells of the monocyte-macrophage lineage. In this study, we examined the effect of MIP-1␥, a C-C chemokine family member, on receptor activator of NF-␬B ligand (RANKL)-stimulated osteoclast differentiation, survival, and activation. RANKL induced osteoclasts to dramatically increase production of MIP-1␥ and to also express the MIP-1␥ receptor CCR1, but had only minor effects on the related C-C chemokines MIP-1␣ and RANTES. Neutralization of MIP-1␥ with specific Ab reduced RANKL-stimulated osteoclast differentiation by 60– 70%. Mature osteoclasts underwent apoptosis within 24 h after removal of RANKL, as shown by increased caspase 3 activity and Downloaded from DNA fragmentation. Apoptosis was reduced by the addition of exogenous MIP-1␥ or RANKL, both of which increased NF-␬B activation in osteoclasts. Neutralization studies showed that the prosurvival effect of RANKL was in part dependent on its ability to induce MIP-1␥. Finally, osteoclast activation for bone resorption was stimulated by MIP-1␥. Taken together, these results demonstrate that MIP-1␥ plays an important role in the differentiation and survival of osteoclasts, most likely via an autocrine pathway. The Journal of Immunology, 2004, 173: 2084–2090. http://www.jimmunol.org/ steoclasts are bone-resorbing multinucleated giant cells MIP-1␥ is a C-C chemokine family member (8, 9). MIP-1␥ that are derived from hemopoietic precursors of the induces the chemotaxis of CD4ϩ and CD8ϩ T cells and monocytes O monocyte-macrophage lineage. Osteoclastic bone re- in vitro (10), and shows potent suppressive activity on the colony sorption consists of multiple steps, including the differentiation of formation of murine bone marrow (BM) myeloid progenitor cells osteoclast precursors; the fusion of mononuclear cells to form ma- (11). MIP-1␥ mRNA is widely expressed in most tissues of normal ture multinucleated osteoclasts; activation to resorb bone; and fi- mice, except brain (9). nally, the survival of activated osteoclasts (1). Receptor activator Using gene microarrays, we found that MIP-1␥ mRNA expres- of NF-␬B ligand (RANKL),3 also known as TNF-related activa- sion was strongly up-regulated in RANKL-induced osteoclasts, tion-induced cytokine or osteoprotegerin ligand, is a member of suggesting its possible involvement in the regulation of this cell by guest on September 24, 2021 the TNF family and is one of key molecules that regulates both type. In this study, we report studies that indicate an important role osteoclastogenesis and bone resorption (2, 3). RANKL expression for MIP-1␥ in RANKL-induced osteoclast formation, survival, and by osteoblasts as well as by activated T cells has been shown to activation in bone resorption, most likely via an autocrine regulate these processes (3, 4). However, the participation of ad- pathway. ditional factors, including autocrine factors induced by RANKL stimulation, is less well characterized. Materials and Methods Chemokines play an important role in immune and inflamma- Mice and reagents tory responses by inducing the migration and adhesion of leuko- cytes. It has been reported that several chemokines may regulate Three- to 5-wk-old BALB/c mice were obtained from The Jackson Labo- the migration and differentiation of osteoclasts, including MIP-1␣ ratory (Bar Harbor, ME). Mouse rRANKL and mouse rM-CSF were pur- chased from PeproTech (Rocky Hill, NJ). Recombinant mouse MIP-1␥, and IL-8 (5–7). However, the cellular source(s) of these chemo- anti-mouse MIP-1␥ Ab, and control IgG1 Ab were obtained from R&D kines and their role in the overall regulation of bone mass remain Systems (Minneapolis, MN). unclear. Cell culture and differentiation of RAW264.7 and BM cells *Department of Cytokine Biology, Forsyth Institute, Boston, MA 02115; and †De- RAW264.7, a mouse macrophage/monocyte cell line, was purchased from partment of Periodontology, Showa University Dental School, Tokyo, Japan American Type Culture Collection (Manassas, VA) (TIB-71). Cells were cultured in DMEM (JRH Biosciences, Lenexa, KS) supplemented with Received for publication September 10, 2003. Accepted for publication May 10% FBS (Invitrogen Life Technologies, Grand Island, NY), 1.5 g/L so- 17, 2004. dium bicarbonate, and penicillin/streptomycin (Invitrogen Life Technolo- The costs of publication of this article were defrayed in part by the payment of page gies). To generate osteoclasts, RAW264.7 cells were plated in 24-well charges. This article must therefore be hereby marked advertisement in accordance plates at a density of 1 ϫ 104 cells/well. Cells were stimulated with 10 with 18 U.S.C. Section 1734 solely to indicate this fact. ng/ml mouse rRANKL for 5 days. Mouse BM cells were collected from 1 This work was supported by Grant DE-07378 from the National Institute of Dental femora and tibiae, as described (12). Briefly, 3- to 5-wk-old female mice and Craniofacial Research, National Institutes of Health. were killed by cervical dislocation under light ether anesthesia. Femora and 2 Address correspondence and reprint requests to Dr. Philip Stashenko, Department of tibiae were dissected, and BM cells were flushed out and cultured in Cytokine Biology, Forsyth Institute, 140 Fenway, Boston, MA 02115. E-mail address: ␣-MEM (Cambrex, Walkersville, MD) supplemented with 10% FBS, 2.0 [email protected] g/L sodium bicarbonate, and penicillin/streptomycin. BM cells were ϫ 6 3 Abbreviations used in this paper: RANKL, receptor activator of NF-␬B ligand; BM, seeded into 24-well plates at a density of 2 10 cells/well in medium bone marrow; FGF, fibroblast growth factor; TRAP, tartrate-resistant acid supplemented with 20 ng/ml RANKL and 50 ng/ml mouse rM-CSF for 7 phosphatase. days. All cultures were maintained at 37°C in a humidified atmosphere of Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 2085 ␮ 5% CO2 in air, with changes of medium every other day. Osteoclast num- dium. Cells were unstimulated (control), or stimulated with 10 gof bers were evaluated by counting tartrate-resistant acid phosphatase RANKL or MIP-1␥ (2.0, 5.0, 10 ng/ml). After 24 h, the cells were removed (TRAP)-positive giant cells. At culture termination, cells were washed with with 5% sodium hypochlorite, and resorbed areas were visualized using PBS and fixed in 10% formalin for 5 min, followed by ethanol/acetone light microscopy. The size of resorbed areas was quantified using NIH (1:1) for 1 min. Osteoclasts were stained for TRAP in the presence of 0.05 Image. M sodium tartrate (Sigma-Aldrich, St. Louis, MO), napthol AS-MX phos- phate (Sigma-Aldrich) as substrate, and fast red LB salt (Sigma-Aldrich). Preparation of nuclear extracts TRAP-positive multinuclear cells (three or more nuclei/cell) were counted Osteoclasts, generated by RANKL stimulation of RAW264.7 cells for 5 under light microscopy. days, were enriched by a brief trypsinization, which removed most mono- Gene array analysis nuclear cells. Osteoclasts were washed twice with PBS, pH 7.4, followed by suspension in 800 ␮l of ice-cold lysis buffer (mmol/L: HEPES, 10; Two array systems were used to detect differences in gene expression be- KCL, 10; EDTA, 0.1; EGTA, 0.1; DTT, 1.0; PMSF, 1.0; and 10 ␮g/ml tween undifferentiated precursor cells and osteoclasts: the Atlas Mouse 1.2 aprotinin, 10 ␮g/ml pepstatin, and 10 ␮g/ml leupeptin). The collected sam- array (BD Clontech, Palo Alto, CA) and the MG-U74 chip (Affymetrix, ples were incubated on ice for 30 min, vortexed for 30 s after addition of Santa Clara, CA). To enrich for differentiated osteoclasts, RANKL-stim- 50 ␮l of 10% Nonidet P-40, and centrifuged for 10 min at 4°C at 5500 rpm. ulated cultures were briefly trypsinized for 1 min to remove other less The nuclei-containing pellets were suspended in ice-cold buffer (mmol/L: adherent cells. This treatment generated a population of Ͼ80% osteoclasts. HEPES, 20; NaCl, 400; EDTA, 1.0; EGTA, 1.0; DTT, 1.0; PMSF, 1.0; and For analysis of gene expression in osteoclasts, total RNA was isolated 10 ␮g/ml aprotinin, 10 ␮g/ml pepstatin, and 10 ␮g/ml leupeptin), incu- from both undifferentiated cells and purified osteoclasts using TRIzol re- bated on ice for 2 h with frequent mixing, and centrifuged for 10 min at 4°C agent (Invitrogen Life Technologies).