Survival B Ligand-Induced Osteoclast

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

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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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 leukocytes. 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 purchased from PeproTech (Rocky Hill, NJ). Recombinant mouse MIP-1␥, and IL-8 (5Ð7). 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

␮ 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 phosphate (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). Total RNA was subsequently treated at 14,000 rpm. The supernatants were collected as nuclear extract and with DNase I (Ambion, Austin, TX) to remove contaminating genomic stored at Ϫ70¡C. The total protein concentration was determined using a DNA and quantified by spectrophotometry. The Atlas array was hybridized protein assay kit (Pierce, Rockford, IL). Downloaded from to a radioactively labeled mixed cDNA probe obtained by reverse transcription of 4 ␮g of total RNA, according to the manufacturer’s instruc- EMSAs tions. After hybridization, the arrays were washed to remove unbound NF-␬B-binding studies were performed using double-stranded oligonucle- probe and exposed to x-ray film. The level of gene expression was analyzed otides containing an NF-␬B consensus binding site. The oligonucleotides and normalized using NIH Image software. The MG-U74 chip was hy- were end labeled with [32P]ATP using T4 polynucleotide kinase (Promega, bridized to a biotinylated mixed cDNA probe, washed, and stained accord- Madison, WI) and incubated with the nuclear extract for 20 min at room ing to the standard Affymetrix GeneChip protocol. The level of gene ex- temperature. The samples were loaded on a 4% nondenaturating polyacryl- pression was analyzed and normalized using statistical algorithms provided amide gel. After electrophoresis, the gel was dried and exposed to http://www.jimmunol.org/ by Affymetrix. Kodak film. Modulation of RANKL-induced osteoclastogenesis Apoptosis assays ␥ MIP-1 (0.1, 0.5, 2.0 ng/ml) was added to cultures of RAW264.7 and BM Assays for caspase 3 activity and degradation of DNA (TUNEL) were used cells to examine its effect on RANKL-induced osteoclast differentiation. To for detection of osteoclast apoptosis. Following RANKL stimulation of ␮ eliminate possible effects of contaminating LPS, 1 g/ml polymyxin B RAW264.7 cells for 5 days, osteoclasts were extensively washed, and re- (Sigma-Aldrich) was simultaneously added to some cultures. To assess the stimulated with MIP-1␥ or RANKL in the presence/absence of anti- ␥ ␥ effect of endogenous MIP-1 , anti-MIP-1 Ab or IgG1 control Ab (0.5, 5.0 MIP-1␥ Ab. Twenty-four hours later, cells were fixed in 4% paraformal- ␮ g/ml) was added to cultures of RAW264.7 and BM cells stimulated with dehyde, and labeled with the caspase 3 substrate rhodamine 110 (100 ␮M)

RANKL. and 2.4 nM TOTO-3 for nuclear staining (Molecular Probes, Eugene, OR) by guest on September 24, 2021 Measurement of chemokines at 37¡C for 30 min. Cells were washed and viewed using a fluorescence microscope. Apoptotic cells exhibited bright green fluorescence. Chemokines were measured in culture supernatants and cell lysates of Apoptosis was also assessed using TUNEL assays. Osteoclasts were RAW264.7 and BM cells. After supernatants were collected, cells were derived from BM cells stimulated with RANKL and M-CSF for 7 days. washed with PBS and lysed in 500 ␮l of protein extraction buffer (0.5% Cultures were washed, and TUNEL assays were conducted using the In Triton X-100, 50 mM Tris-HCl, 0.3 M NaCl, and 5 mM EDTA). MIP-1␥, Situ Cell Death Detection kit, tetra-methyl-rhodamine red (Roche Applied MIP-1␣, and RANTES levels were determined using commercially avail- Science, Indianapolis, IN), according to the manufacturer’s protocols. Ap- able ELISA kits (R&D Systems). optotic cells were identified by bright red fluorescence in this assay. Semiquantitative RT-PCR Statistical analysis Semiquantitative RT-PCR was used to examine CCR1 gene expression in In all studies, differences between groups were analyzed using Student’s t undifferentiated cells and purified osteoclasts. For this, 1 ␮g of total RNA test with the Bonferroni correction for multiple comparisons. was reverse transcribed using Superscript II (Invitrogen Life Technologies) and random primers, according to the manufacturer’s instructions. cDNA Results was subjected to PCR amplification with Taq polymerase (Qiagen, Valen- Induction of MIP-1␥ expression by RANKL during Ј cia, CA) using specific mouse CCR1 primers: sense, 5 -gtgttcatcattggagt osteoclastogenesis ggtgg-3Ј; antisense, 5Ј-ggttgaacaggtagatgctggtc-3Ј (13). Osteoclast formation was induced by RANKL stimulation of Evaluation of osteoclast survival and activity RAW264.7 monocytes for 5 days, or by stimulation of normal For cell survival analysis, osteoclasts were generated from RAW264.7 mouse BM cells for 7 days with M-CSF and RANKL. As previ- cells for 5 days. Adherent osteoclasts were washed extensively with PBS ously reported, RANKL-stimulated RAW264.7 and BM cells dif- to completely remove RANKL. Cells were subsequently cultured without or with 10 ng/ml RANKL in the presence/absence of 2.0 ng/ml MIP-1␥. ferentiated into TRAP-positive osteoclasts that expressed high lev- Neutralizing anti-MIP-1␥ Ab (5 ␮g/ml) or control IgG1 Ab (5 ␮g/ml) was els of the osteoclast markers TRAP, cathepsin K, and the proton simultaneously added with RANKL to determine the role of MIP-1␥ in pump subunit ATP6I, and produced resorption pits on bone slices apoptosis of RANKL-induced osteoclasts. After 24, 48, and 72 h, survival and calcium phosphate-coated slides (14). was determined by counting adherent TRAP-positive osteoclasts. Two gene array systems (Atlas and Affychip) were used to study To determine the effect of MIP-1␥ on bone-resorbing activity, osteoclasts were generated in three-dimensional collagen gels (Chemicon In- gene expression following RANKL induction of osteoclast forma- ternational, Temecula, CA). Dishes (60 mm) were covered with a collagen tion. Total RNA was extracted from RANKL-induced osteoclasts, gel solution prepared according to the manufacturer’s instructions. and was used as a template to generate mixed cDNA probes. In the RAW264.7 cells were seeded onto the gels and cultured with 10 ng/ml Atlas system (1100 genes), we observed a highly significant up- RANKL for 5 days. Cells were removed following digestion of gels with ␥ 1000 U/ml collagenase (Sigma-Aldrich) at 37¡C for 30 min. Aliquots of regulation of mRNA for the chemokine MIP-1 in RANKL-stim- the harvested cell suspension were seeded onto submicron calcium phos- ulated osteoclasts derived from RAW264.7 cells, compared with phate films (Osteologic; BD Biosciences, Bedford, MA) in 250 ␮lofme- unstimulated precursor cells (Table I). This result was confirmed 2086 MIP-1␥ IN OSTEOCLASTOGENESIS

Table I. Array analysis of MIP-1␥ gene expression in osteoclasts

Array Differentiated Osteoclasts/Undifferentiated Cells

Atlas RAW264.7 5.54a Affymetrix RAW264.7 Normal bone marrow MG-U74 chips 2.28a,b 1.89a,b

a Ratio of intensity of gene expression, normalized to housekeeping gene GAPDH. b p Ͻ 0.000001. Wilcoxon signed rank test. and extended using the Affymetrix system (32,000 genes) and mRNA derived from osteoclasts induced from normal BM as well as RAW264.7 cells (Table I). The induction of MIP-1␥ was more FIGURE 2. CCR1 expression in RANKL-induced osteoclasts. Oste- significant than any other chemokine or cytokine gene represented oclasts were induced by RANKL from RAW264.7 and BM cells. mRNA specific for CCR1 was amplified by semiquantitative RT-PCR for CCR1 on these arrays, suggesting a possible role in osteoclast develop- and GAPDH as a control. PCR was performed under conditions determined ment and/or function. to be in the linear range of product formation. Downloaded from Expression of C-C chemokines in developing osteoclasts MIP-1␥ binds to CCR1, which is also activated by related C-C els. Interestingly, the levels of MIP-1␣ in supernatants peaked by chemokines MIP-1␣ and RANTES. We therefore examined the day 2, although cell-associated levels continued to increase to day production of MIP-1␥, MIP-1␣, and RANTES proteins during the 5. RANTES was also weakly induced by RANKL, but its levels process of RANKL-induced osteoclastogenesis from RAW264.7 were only 1Ð2% of those of MIP-1␥ at any time point (maximum: cells. As shown in Fig. 1A, RANKL strongly stimulated the pro- 50 pg/ml). http://www.jimmunol.org/ duction of MIP-1␥ in both cell lysates and culture supernatants MIP-1␥ was similarly induced following RANKL stimulation of over the 5-day culture period. MIP-1␣ was also induced by normal BM cells (Fig. 1B). However, compared with RAW264.7 RANKL, as previously reported (15), albeit at 10-fold lower lev- by guest on September 24, 2021

FIGURE 3. Anti-MIP-1␥ Ab reduces RANKL-induced osteoclast differentiation. A, RAW264.7 cells were cultured with RANKL (10 ng/ml) and neutralizing anti-MIP-1␥ Ab or an unreactive control IgG1 Ab for 5 FIGURE 1. Kinetics of chemokine production by RANKL-stimulated days. B, BM cells were cultured in the presence of RANKL (20 ng/ml) and RAW264.7 and BM cells. A, RAW264.7 cells were stimulated with RANKL M-CSF (50 ng/ml), in the presence of neutralizing anti-MIP-1␥ Ab or (10 ng/ml) for 5 days. B, BM cells were stimulated with RANKL (20 ng/ml) unreactive control IgG1 Ab for 7 days. TRAP-positive cells with more than and M-CSF (50 ng/ml) for 7 days. Chemokine levels were measured by three nuclei were counted as osteoclasts. The results shown are the mean Ϯ .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .ELISA. Results represent the mean Ϯ SD of triplicate cultures. SD of three independent experiments The Journal of Immunology 2087 Downloaded from

FIGURE 5. MIP-1␥ enhances osteoclast survival. RAW264.7 cells were stimulated with RANKL (10 ng/ml) for 5 days, extensively washed, and subsequently stimulated with RANKL (10 ng/ml), MIP-1␥ (2 ng/ml), or medium as a control. Neutralizing anti-MIP-1␥ Ab (5 ␮g/ml) or control ␮ IgG1 Ab (5 g/ml) was simultaneously added with RANKL to determine http://www.jimmunol.org/ the role of MIP-1␥ in RANKL-stimulated osteoclast survival. TRAP-positive cells with more than three nuclei were counted as osteoclasts. The p Ͻ ,ء .FIGURE 4. Effect of exogenous MIP-1␥ on RANKL-induced osteoclast results shown are the mean Ϯ SD of three independent experiments differentiation. A, RAW264.7 cells were stimulated for 5 days with the 0.05 RANKL ϩ control Ab vs RANKL ϩ anti-MIP-1␥. indicated doses of MIP-1␥, in the presence or absence of RANKL (10 ng/ml). B, BM cells were stimulated for 7 days with MIP-1␥ in the presence or absence of RANKL (20 ng/ml) and M-CSF (50 ng/ml). The culture ﬁndings were replicated in two additional experiments (data not medium was supplemented with (f) or without (Ⅺ)1␮g/ml polymyxin B shown). to block any contaminating LPS. TRAP-positive cells with more than three The effect of adding exogenous rMIP-1␥ on osteoclast forma- nuclei were counted as osteoclasts. The results shown are the mean Ϯ SD tion was also determined. As seen in Fig. 4, TRAP-positive oste- by guest on September 24, 2021 of three independent experiments. oclasts were not induced by MIP-1␥ alone, nor did exogenous MIP-1␥ have a synergistic effect with RANKL on osteoclastogenesis in either cell system. These results are perhaps not unexpected, given the high level of endogenous MIP-1␥ production. Neverthe- cells, RANTES was expressed initially at somewhat higher levels, but less, they indicate that endogenous MIP-1␥ increases RANKL- expression declined with increasing times after culture induction, and induced osteoclast formation, but has no independent ability to MIP-1␣ was nearly undetectable. Taken together, these results sug- induce osteoclastogenesis. gest that MIP-1␥ is the predominant C-C chemokine produced by RANKL-stimulated precursor cells during osteoclastogenesis. Effect of MIP-1␥ on osteoclast survival It has been reported that mature osteoclasts rapidly undergo apo- CCR1 expression by osteoclasts ptosis in the absence of bone-resorptive stimuli such as RANKL, We next examined RAW264.7 and BM cells for the expression of LPS, or IL-1␣ (16). We therefore determined whether MIP-1␥ CCR1 mRNA upon RANKL stimulation. As shown in Fig. 2, might also play a role in maintaining osteoclast viability. In these CCR1 mRNA was undetectable in unstimulated RAW264.7 cells, experiments, RANKL was removed from cultures of differentiated but was present at low levels in unstimulated BM cells. RANKL osteoclasts by extensive washing of the cells on day 5. Cells were stimulation strongly induced CCR1 mRNA in RAW264.7 and, to then recultured for an additional 24Ð72 h in the presence or ab- a lesser extent, BM cells. Taken together, these results show that sence of MIP-1␥ or RANKL as a positive control. As shown in RANKL induces both MIP-1␥ and its receptor during osteoclast Fig. 5, the number of surviving osteoclasts was reduced by 90% differentiation, suggesting the operation of an autocrine pathway. after 24 h in the absence of a stimulating agent (medium alone). RANKL restimulation promoted osteoclast survival, as indicated ␥ Modulation of osteoclastogenesis by MIP-1 by only a 30% reduction in viable TRAP-positive cells after 24 h. We then investigated the role of endogenously produced MIP-1␥ Interestingly, the addition of MIP-1␥ alone also prevented cell in RANKL-induced osteoclastogenesis in RAW264.7 and normal death, albeit somewhat less effectively than RANKL itself. We BM cells. A neutralizing anti-MIP-1␥ Ab was added to cultures also determined whether the survival-promoting effect of RANKL beginning on day 0, and was replenished periodically throughout was dependent on its ability to induce MIP-1␥ expression. As in- the osteoclast induction period. As seen in Fig. 3, in both cell dicated (Fig. 5), the prosurvival activity of RANKL was reduced systems, the addition of anti-MIP-1␥ Ab resulted in a decreased by ϳ60% in the presence of anti-MIP-1␥ Ab, whereas an isotype- number of TRAP-positive osteoclasts, relative to control IgG1 Ab. matched control IgG1 Ab had no effect. The reduction was ϳ60% in cultures treated with 5 ␮g/ml anti- That the loss of osteoclasts was the result of apoptosis rather MIP-1␥ Ab, and 45% in cultures treated with 0.5 ␮g/ml Ab. These than necrosis was demonstrated using a ﬂuorescence-based assay 2088 MIP-1␥ IN OSTEOCLASTOGENESIS Downloaded from http://www.jimmunol.org/

FIGURE 6. A, Antiapoptotic effect of MIP-1␥ on mature osteoclasts as assessed by caspase 3 staining. RANKL-induced RAW264.7-derived osteoclasts were washed and recultured with: 1) medium, 2) RANKL, 3) MIP-1␥, 4) RANKL ϩ anti-MIP-1␥ Ab (5 ␮g/ml), or 5) RANKL ϩ control IgG1 Ab (5 ␮g/ml) for 24 h. Cell staining with rhodamine 110 (green) indicates caspase 3 activity; TOTO-3 (blue) counterstain for nuclei. Arrowheads: osteoclasts. Magniﬁcation: ϫ100. B, Antiapoptotic effect of MIP-1␥ on BM cell-derived mature osteoclasts, as assessed by TUNEL assay. RANKL ϩ M-CSF-induced BM cell-derived osteoclasts were washed and recultured with: 1) medium, 2) RANKL, 3) MIP-1␥, 4) RANKL ϩ anti MIP-1␥ (5 ␮g/ml), or 5) RANKL ϩ control

IgG1 Ab (5 ␮g/ml) for 24 h. Apoptotic cells were detected by TUNEL with tetra-methyl-rhodamine red (red). Arrowheads indicate outline of a representative by guest on September 24, 2021 osteoclast. C, Effect of MIP-1␥ on NF-␬B activation in osteoclasts. Osteoclasts were generated by RANKL stimulation of RAW264.7 cells for 5 days, washed, and restimulated with RANKL (10 ng/ml) (lane 4), MIP-1␥ (10 ng/ml) (lane 5), or medium (lane 3) as a control for 12 h. Nuclear extracts were assessed for NF-␬B activity by EMSA. Positive control: day 5 RANKL-stimulated osteoclasts (lane 2); negative control; no nuclear extract (lane 1).

for the specific activity of caspase 3 (Fig. 6A). Osteoclasts cultured were cultured in three-dimensional collagen gels, isolated by en- in the presence of RANKL exhibited minimal apoptosis ( panel 2), zymatic digestion, and replated onto submicron calcium phosphate compared with cells from which RANKL was removed ( panel 1). films for an additional 24 h in the presence/absence of MIP-1␥ or Cells cultured with MIP-1␥ alone were partially protected from RANKL as a positive control. As shown in Fig. 7, osteoclasts were apoptosis ( panel 3). The prosurvival effect of RANKL was again stimulated to form numerous resorption pits in the presence of demonstrated to be partially dependent on its ability to induce added RANKL, but not in its absence. Of interest, the addition of MIP-1␥, as shown by anti-MIP-1␥ Ab blockade of the protective MIP-1␥ alone also resulted in a marked stimulation of resorption, effect of RANKL ( panel 4 vs 5). These results were further con- to a level similar to that seen with RANKL-stimulated cells. These firmed in similar studies using osteoclasts derived from normal results suggest that MIP-1␥ stimulates the activation as well as the BM cells and TUNEL assays (Fig. 6B). survival of mature osteoclasts. Given that many of the factors that promote osteoclast survival, including RANKL, act by stimulating NF-␬B, we examined the effect of MIP-1␥ on this transcription factor. As shown in Fig. 6C, Discussion following extensive washing of mature osteoclasts, RANKL re- It is increasingly evident that extensive cross talk occurs between stimulation strongly induced NF-␬B DNA-binding activity in ma- the immune and skeletal systems. In particular, the differentiation ture osteoclasts, as assessed by EMSA. MIP-1␥ by itself also stim- and activity of osteoclasts, and hence bone mass, can be modulated ulated NF-␬B, but less strongly than RANKL, which correlated by an ever-expanding number of cytokines/chemokines, many of with the level of its prosurvival activity (Fig. 6, A and B). Taken which derive from immune cells. In the present investigation, we together, these results support the conclusion that a primary func- used gene arrays to identify mediators that were up-regulated fol- tion of MIP-1␥ may be to promote the survival of mature oste- lowing RANKL stimulation of osteoclast precursor cells. Our data oclasts by preventing apoptosis. show that of all the mediators screened, MIP-1␥ was the most strongly up-regulated in osteoclasts derived from RANKL-stimu- ␥ Effect of MIP-1 on bone-resorbing activity lated monocytes/macrophages. CCR1, the high affinity receptor for Finally, we determined the effect of MIP-1␥ on the bone-resorbing MIP-1␥, was also increased following RANKL stimulation. Inhi- activity of mature osteoclasts. RANKL-induced RAW264.7 cells bition of MIP-1␥ resulted in decreased osteoclast formation and The Journal of Immunology 2089

ization of MIP-1␥ reduced osteoclast formation by ϳ60%, it remains possible that MIP-1␣ and RANTES could also participate in RANKL-induced osteoclastogenesis following interaction with CCR1, albeit with a more modest effect. Alternatively, residual MIP-1␥-independent RANKL-induced osteoclast formation may proceed via pathways independent of these chemokines. Most of our ﬁndings with RAW264.7 cells were replicated using normal BM cells, conﬁrming the role of MIP-1␥ in a nontrans- formed osteoclast precursor (i.e., macrophage) population. At the same time, a concern in using BM cells is that they are heteroge- neous and contain a number of cell types that may express RANKL following activation, including T cells, B cells, and osteoblasts (2, 20Ð22). We examined the phenotype of nonosteoclastic cells after 7 days’ culture, and found that T cells (CD3ϩ, 0.7%), B cells (CD19ϩ, 0.5%), and osteoblasts/stromal cells (alkaline phosphatase positive, Ͻ0.5%) constituted only minor components after 7 days in culture (H. Sasaki, unpublished observations). Monocytes (CD14ϩ), which are the precursors of osteoclasts,

comprised most of the remaining nonosteoclastic cells. Thus, al- Downloaded from though these contaminating populations may have the capacity to express RANKL and modulate the observed MIP-1␥ response, we believe that this effect is minimal given the small numbers of such cells and the correspondence in ﬁndings between the two cell systems.

FIGURE 7. MIP-1␥ enhances the activation of preformed osteoclasts. Osteoclasts rapidly undergo apoptosis unless stimulated by ex- http://www.jimmunol.org/ RAW264.7-derived osteoclasts recovered from collagen gel cultures, and ogenous mediators, which may include M-CSF, RANKL, IL-1, were plated on Osteologic Multitest Slides to assess resorptive activity fibroblast growth factor 2 (FGF2), or LPS (16, 23Ð26). With the without/with RANKL (10 ng/ml) or MIP-1␥ (2, 5, and 10 ng/ml). A, Pho- exception of M-CSF and FGF2, these survival-promoting stimuli ϫ tomicrograph of resorption areas visualized by light microscopy ( 10). B, act by inducing NF-␬B (26, 27), which is well established as an The size of resorbed areas was quantified using NIH Image. Data represent antiapoptogen (28, 29). FGF2, which directly stimulates activation the mean Ϯ SD of triplicate cultures. and survival of mature osteoclasts, mediates its effects through p42/p44 MAPK. We confirmed that MIP-1␥, like RANKL, stimulated NF-␬B activity, and furthermore demonstrated that the pro- reduced resorptive activity. Furthermore, MIP-1␥ promoted oste- survival activity of RANKL was partially dependent on its ability by guest on September 24, 2021 oclast survival and prevented apoptosis, and was responsible for a to induce MIP-1␥. Of interest, MIP-1␥ also stimulated the activa- major proportion of the prosurvival activity of RANKL itself. To tion of mature osteoclasts, although it is difficult to separate this our knowledge, this is the first report that MIP-1␥ may play an effect from its ability to promote cell viability. Thus, in contrast to important role in regulating osteoclastic bone resorption, via ef- most survival factors that act in a paracine manner, our results fects on cell differentiation, survival, and activation. suggest that osteoclasts protect themselves from apoptosis through MIP-1␥ is a relatively recently described C-C chemokine with a production of MIP-1␥ as an autocrine survival factor. At present, predicted length of 100 aa, which is identical with CCF18 (8). the signal transduction pathways involved in MIP-1␥-induced os- MIP-1␥ is constitutively expressed by a wide variety of tissues (9), teoclast survival via CCR1 have not been characterized. and exclusively binds to CCR1 on mouse neutrophils in vitro (9, In conclusion, our data indicate that MIP-1␥ represents a new 11). MIP-1␥ was recently reported to be increased in rat BM cells and potentially important factor in the bone microenvironment that stimulated with RANKL, although its function was not determined regulates osteoclastic bone resorption. The role that MIP-1␥ may (17). Two other C-C chemokines, MIP-1␣ and RANTES, also bind play in both normal bone turnover and osteolytic diseases remains to CCR1 (18), and are therefore potentially able to modulate os- to be established. teoclast development and function. However, we found that MIP-1␥ was by far the predominant chemokine produced by References RANKL-stimulated RAW264.7 and BM cultures, compared with 1. Reddy, S. V., and G. D. Roodman. 1998. Control of osteoclast differentiation. relatively minor amounts of MIP-1␣ and RANTES. Additionally, Crit. Rev. Eukaryotic Gene Expression 8:1. ␣ 2. Suda, T., N. Takahashi, N. Udagawa, E. Jimi, M. T. Gillespie, and T. J. Martin. the function of MIP-1 and RANTES in osteoclasts is somewhat 1999. 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