Reciprocal cross-talk between RANKL and interferon-╨Ю╤Цinducible protein 10 is responsible for bone-erosive experimental arthritis.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 58, No. 5, May 2008, pp 1332–1342 DOI 10.1002/art.23372 © 2008, American College of Rheumatology Reciprocal Cross-Talk Between RANKL and Interferon-␥–Inducible Protein 10 Is Responsible for Bone-Erosive Experimental Arthritis Han Bok Kwak,1 Hyunil Ha,2 Ha-Neui Kim,2 Jong-Ho Lee,2 Hun Soo Kim,1 Seungbok Lee,2 Hyun-Man Kim,2 Jung Yeon Kim,3 Hong-Hee Kim,2 Yeong Wook Song,3 and Zang Hee Lee2 Objective. Interferon-␥–inducible protein 10 (IP10; also called CXCL10), a chemokine important in the migration and proliferation of T cells, is induced in a wide variety of cell types. However, the role of IP-10 in rheumatoid arthritis (RA) remains largely unknown. The purpose of this study was to examine the potential role of IP-10 in bone resorption and RA through examination of a mouse model of collagen-induced arthritis (CIA). Methods. The effects of IP-10 on mouse T cells during osteoclast differentiation were examined in migration assays. The bone-erosive activity of IP-10 was determined in vivo in a mouse model of CIA by histologic and immunostaining analyses. Cytokine levels in serum and culture medium were measured with sandwich enzyme-linked immunosorbent assays. Results. Serum concentrations of IP-10 were sig- nificantly higher in mice with CIA than in control mice. RANKL greatly induced IP-10 expression in osteoclast precursors, but not in mature osteoclasts. IP-10 stimulated the expression of RANKL and tumor necrosis factor ␣ (TNF␣) in CD4ⴙ T cells and induced osteoclastogenesis in cocultures of CD4ⴙ T cells and osteoclast precursors. However, IP-10 did not induce RANKL or TNF␣ in CD8ⴙ T cells. Treatment with neutralizing antibody to IP-10 significantly inhibited the infiltration of CD4ⴙ T cells and F4/80ⴙ macrophages into the synovium and attenuated bone destruction in mice with CIA. Furthermore, levels of RANKL and TNF␣ were inhibited by antibody to IP-10. Bone erosion was observed in mice infected with an IP-10 retrovirus. Conclusion. Our findings suggest that IP-10 plays a critical role in the infiltration of CD4ⴙ T cells and F4/80ⴙ macrophages into inflamed joints and causes bone destruction. Our results provide the first evidence that IP-10 contributes to the recruitment of inflammatory cells and is involved in bone erosion in inflamed joints. Supported by the Ministry of Science and Technology of Korea through grants from the Center for Biological Modulators, the Stem Cell Research Center of the 21st Century Frontier Research and Development Program, and the National Research Laboratory Program for Rheumatic Disease. 1 Han Bok Kwak, PhD, Hun Soo Kim, MD: Wonkwang University School of Medicine, Iksan, Republic of Korea; 2Hyunil Ha, PhD, Ha-Neui Kim, BS, Jong-Ho Lee, BS, Seungbok Lee, PhD, Hyun-Man Kim, DDS, PhD, Hong-Hee Kim, PhD, Zang Hee Lee, DDS, PhD: Seoul National University School of Dentistry, Seoul, Republic of Korea; 3Jung Yeon Kim, MS, Yeong Wook Song, MD, PhD: Seoul National University College of Medicine, Seoul, Republic of Korea. Address correspondence and reprint requests to Yeong Wook Song, MD, PhD, Department of Internal Medicine, Medical Research Center, Seoul National University College of Medicine, 28 YeongonDong, Jongro-Gu, Seoul 110-749, Republic of Korea (e-mail: firstname.lastname@example.org); or to Zang Hee Lee, DDS, PhD, Department of Cell and Developmental Biology, Dental Research Institute, Seoul National University College of Dentistry, 28 Yeongon-Dong, Jongro-Gu, Seoul 110-749, Republic of Korea (e-mail: email@example.com). Submitted for publication November 29, 2006; accepted in revised form January 4, 2008. The regulation of osteoclasts is vital for maintaining balance in bone remodeling (i.e., bone resorption by osteoclasts and bone formation by osteoblasts) and is thus important in the treatment of bone disease. Boneresorbing osteoclasts are derived from hematopoietic cells of the monocyte/macrophage lineage, and they differentiate into multinucleated cells through multiple processes (1). Osteoclast formation and activity are regulated by local factors and by stromal and osteoblast cells in the bone environment (2). Increases in osteoclast number and activity can be caused by systemic alterations, such as the up-regulation of osteotropic or oste1332 IP-10 MEDIATION OF BONE-EROSIVE EXPERIMENTAL ARTHRITIS oclastogenic factors or a deficiency of estrogen, and in turn, cause bone disease, including rheumatoid arthritis (RA), periodontal disease, and osteoporosis (3). In particular, RANKL, a member of the tumor necrosis factor (TNF) family that is expressed on stromal and osteoblast cells, plays an essential role in osteoclast differentiation and function. Several inflammatory cytokines, including TNF␣ and interleukin-1 (IL-1), can induce RANKL expression on stromal and osteoblast cells, which plays an important role in bone and cartilage destruction in RA (4). Thus, the regulation of RANKL expression is important for preventing bone disorders caused by increased osteoclast formation. Chemokines are a superfamily of cytokines that are important in inflammation and immune responses. Chemokines can be divided into 4 main groups (C, CC, CXC, and CX3C) according to the presence of none, 1, or 3 amino acids between the first 2 cysteine residues, respectively (5). Several chemokines promote bone resorption by inducing osteoclast formation and survival, as well as by directly inducing the migration and adhesion of leukocytes (6). In particular, the expression of macrophage inflammatory protein 1␣ (MIP-1␣) and MIP-1␤ in cells from patients with multiple myeloma enhances osteolytic lesions by enhancing osteoclast formation and bone resorption (7). Interferon-␥–inducible protein 10 (IP-10; also called CXCL10) was initially identified as a chemokine induced by interferon-␥ (IFN␥) that is secreted by various cell types (8). IFNstimulated response element and B sites in the IP-10 promoter are important for IFN␥-induced expression (9). IP-10 binds the receptor CXCR3 and regulates immune responses through the activation and recruitment of leukocytes, including T cells, eosinophils, and monocytes (10,11). Also, IP-10 has antitumor activity in vivo, which has been attributed to the recruitment of lymphocytes. However, the role of IP-10 in bone resorption has not yet been reported. RA, a chronic inflammatory disease, is characterized by excessive bone resorption in inflamed joints that is initially promoted through the recruitment of activated T cells (12), a distinct subset of which is Th17 cells, that mediate osteoclastogenesis and, in turn, induce bone erosion in the RA synovium (13). Thus, the regulation of infiltrated T cells into the synovium is important for the progression of RA. Although it has been reported that many chemokines and inflammatory cytokines induce the infiltration of inflammatory cells (chiefly, mononuclear cells and T cells) into the synovium in inflamed joints and mediate inflammation (14,15), the etiology of RA remains unknown. IP-10– 1333 deficient mice do not respond to allogeneic or antigenic stimulation and have defective trafficking of T cells (16). IP-10 is expressed in many T cell–related inflammatory diseases, such as multiple sclerosis, atherosclerosis, and lichen planus (17–19). Furthermore, a recent study by Hanaoka et al (20) showed that IP-10 is elevated in the synovial fluids of RA patients. These results suggest that IP-10 plays an important role in T cell–related inflammation and in the recruitment of T cells to sites of inflammation. In the present study, we examined a potential role of IP-10 in bone resorption and RA progression by using a mouse model of collagen-induced arthritis (CIA). MATERIALS AND METHODS Reagents. Recombinant human RANKL, macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), mouse IP-10, and anti–IP-10 antibody were obtained from PeproTech (London, UK). Antibodies against phospho-ERK, ERK, phospho-Akt, Akt, phospho-p38, and p38 were from Cell Signaling Technology (Beverly, MA). Antibodies against nuclear factor of activated T cells c1 (NF-ATc1), NF-ATc2, and actin were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse IgG was from R&D Systems (Minneapolis, MN). Antibodies against CD4, IP-10 (R&D Systems), and F4/80 (eBioscience, San Diego, CA) were also used for immunohistochemistry experiments. Mouse strains. DBA/1 mice were purchased from Samtako Bio Korea (Osan, Republic of Korea) and were used for the induction of CIA. The ICR mouse strain was used for other animal experiments. Mice were housed under specific pathogen–free conditions, and all animal experiments were performed under a protocol approved by the Institute Committee of Seoul National University. Induction and assessment of CIA. Type II collagen (Chondrex, Seattle, WA) was dissolved in 50 mM acetic acid at 4°C (2 mg/ml) and was emulsified with an equal volume of Freund’s complete adjuvant (Chondrex) containing 2 mg/ml of Mycobacterium tuberculosis. DBA/1 mice were injected intradermally at the base of the tail with 150 l of the emulsion. After 14 days, type II collagen was emulsified with Freund’s incomplete adjuvant (Chondrex) and injected as described above. The severity of arthritis was assessed according to paw swelling and was scored on a scale of 0–3, where 0 ⫽ normal, 1 ⫽ swelling of the toes, 2 ⫽ swelling of the sole of the foot or increased swelling, and 3 ⫽ severe swelling or swelling of the entire paw. On day 42, a radiograph was taken with a microfocal computed tomography apparatus. The bone erosion values of each sample were measured from the radiographic images by using a SkyScan 1072 Micro-CT system (SkyScan, Kontich, Belgium). Paws were removed, fixed, decalcified, and sectioned, and serum was obtained for enzyme-linked immunosorbent assays (ELISAs). Treatment with anti–IP-10 antibody. After the first immunization with type II collagen (day 14), mice were given 1334 a booster injection of type II collagen and were also given an intravenous injection of control IgG or anti–IP-10 antibody (200 g/mouse) into the tail vein. On day 42, paws and sera were collected for histologic assessments and ELISAs, respectively. Histologic and immunostaining analyses. Tissues were removed and fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for 1 day at 4°C and were then decalcified in 12% EDTA. Decalcified bones were paraffin-embedded and sectioned. For histologic examination, sections were stained with hematoxylin and eosin. For immunostaining experiments, sections were dewaxed with xylene and then dehydrated with ethanol. After nonspecific binding was blocked with 2% bovine serum albumin (BSA), the sections were incubated for 1 hour with the primary antibodies in phosphate buffered saline (PBS) containing 2% BSA. Sections were washed 3 times with PBS and then incubated with the appropriate secondary antibodies (1:5,000 dilution). Finally, the sections were examined under a confocal microscope. The immunostained cells were quantified by using the Image Pro-Plus program, version 4.0 (Media Cybernetics, Silver Spring, MD). Isolation of osteoclast precursors and activated CD4ⴙ T cells. Osteoclast precursors and mature osteoclasts were prepared according to routine methods. Briefly, bone marrow cells were obtained from the long bones, suspended in ␣-minimum essential medium (␣-MEM; Welgene, Daegu, Republic of Korea) containing 10% fetal bovine serum (FBS) in the presence of M-CSF (10 ng/ml), and cultured for 1 day. Nonadherent cells were then cultured for 3 days in the presence of M-CSF (30 ng/ml), and adherent cells were used as osteoclast precursors. Mature osteoclasts were generated by cocultures of bone marrow cells and calvarial osteoblasts. CD4⫹ T cells were isolated from mouse spleens. Spleens were mashed in Hanks’ balanced salt solution containing 3⫻ antibiotics. Cells were harvested, and red blood cells were removed. The remaining cells were collected and separated with a Ficoll-Histopaque (Sigma-Aldrich) discontinuous gradient. The interface containing the cells was washed with PBS, resuspended in RPMI 1640 (Gibco BRL, Grand Island, NY) containing 10% FBS, and then cultured for 1 day. Nonadherent cells were then cultured for 4 days in the presence of IL-2 (50 ng/ml). Activated CD4⫹ and CD8⫹ T cells were purified by positive selection by using anti-CD4 or anti-CD8 magnetic beads, respectively. The purity of CD4⫹ and CD8⫹ T cells was ⱖ95%, as assessed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA). Western blotting. Cells were lysed in lysis buffer (50 mM Tris Cl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM NaF, 1 mM Na3VO4, and 1% sodium deoxycholate) containing protease inhibitors. Equal amounts of protein were subjected to electrophoresis on 10–15% sodium dodecyl sulfate–polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and subjected to Western blotting with antibodies to IP-10, NF-ATc1, NF-ATc2, phospho-ERK, ERK, phospho-Akt, Akt, phospho-p38, p38, and actin. Reverse transcription–polymerase chain reaction (RTPCR) analysis. For RT-PCR assays, total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s recommendations. Two micrograms of total RNA from each sample was reverse transcribed with Superscript II reverse transcriptase (Invitrogen). The primers used were as KWAK ET AL follows: for RANKL, 5⬘-CAGGTTTGCAGGACTCGAC-3⬘ (sense) and 5⬘-AGCAGGGAAGGGTTGGACA-3⬘ (antisense); for TNF␣, 5⬘-ACACCGTCAGCCGATTTGC-3⬘ (sense) and 5⬘-CCCTGAGCCATAATCCCCTTT-3⬘ (antisense); for IFN␥, 5⬘-GTTTGAGGTCAACAACCCAC-3⬘ (sense) and 5⬘AATCTGAGTTCAGTCAGCCG-3⬘ (antisense); for CXCR3, 5⬘-GCCACCCATTGCCAGTACAAC-3⬘ (sense) and 5⬘TCCCACAAAGGCATAGAGCAGC-3⬘ (antisense); for IP-10, 5⬘-AAGCCTCCCCATCAGCACCA-3⬘ (sense) and 5⬘TGTCCATCCATCGCAGCACC-3⬘ (antisense); for cyclooxygenase 2 (COX-2), 5⬘-TCAGCCAGGCAGCAAATCCTTG-3⬘ (sense) and 5⬘-TAGTCTCTCCTATGAGTATGAGT-3⬘ (antisense); for IL-1␤, 5⬘-GACCTTCCAGGATGAGGACA-3⬘ (sense) and 5⬘-GATTCTTTCCTTTGAGGCCC-3⬘ (antisense); for IL-17, 5⬘-GAAGGCCCTCAGACTACCTC-3⬘ (sense) and 5⬘-TCTTCTCGACCCTGAAAGTG-3⬘ (antisense); and for GAPDH, 5⬘-CAAGGCTGTGGGCAAGGTCA-3⬘ (sense) and 5⬘-AGGTGGAAGAGTGGGAGTTGCTG-3⬘ (antisense). The amplified PCR products were separated on a 1% agarose gel. Densitometry values for each band were quantified with the use of the Image Pro-Plus program, version 4.0. Cytokine ELISAs. Concentrations of cytokines in serum and culture medium were measured with sandwich ELISA kits (R&D Systems) according to the manufacturer’s instructions. Assessment of osteoclastogenesis. Osteoclast precursors and CD4⫹ cells were cocultured for 6 days at 37°C in 48-well plates in the presence of M-CSF (10 ng/ml) and IP-10 (100–200 ng/ml). Cells were fixed with 3.7% formalin, permeabilized in 0.1% Triton X-100, and stained by the addition of tartrate-resistant acid phosphatase (TRAP) solution (SigmaAldrich). Migration assay. The migration of CD4⫹ T cells and osteoclast precursors was evaluated with the use of a Boyden chamber (24-well, 3-m or 8-m pore size membrane; Corning Costar, Cambridge, MA), as previously described (21). Preparation of retroviruses. The coding sequence for IP-10 was cloned by PCR techniques using a pair of primers for the mouse IP-10 gene and was inserted into the pMX-IRESEGFP vector. Plat E cells were plated in 10-cm dishes and were transiently transfected with pMX-IRES-EGFP DNA or with pMX-IP-10-IRES-EGFP DNA by using Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer. Forty-eight hours after transfection, the culture medium was collected and used as a retrovirus. Administration of IP-10 to cells and to mice. Osteoclast precursors were infected with control or IP-10 retrovirus in the presence of M-CSF (30 ng/ml) and Polybrene (6 g/ml) for 8 hours. The medium was replaced with fresh ␣-MEM containing 10% FBS, antibiotics, and M-CSF (30 ng/ml) and was further incubated for 2 days. Green fluorescent protein (GFP)–expressing cells were examined with confocal microscopy. For in vivo study, control or IP-10 retrovirus (50 l) was injected weekly for 6 weeks into the tibial metaphysis of ICR mice. A radiograph of each mouse was taken with a soft x-ray machine. Statistical analysis. Each experiment was performed in triplicate and was repeated at least twice. All quantitative data are presented as the mean ⫾ SD. Statistical differences were analyzed by one-way analysis of variance followed by the Bonferroni test. IP-10 MEDIATION OF BONE-EROSIVE EXPERIMENTAL ARTHRITIS Figure 1. Expression of interferon-␥–inducible protein 10 (IP-10) in collagen-induced arthritis (CIA). A, Histologic features of the hind paws of control mice and mice with CIA. Type II collagen was emulsified with Freund’s complete adjuvant, and DBA/1 mice (n ⫽ 5) were injected intradermally at the base of the tail with 150 l of the emulsion on days 0 and 14. Mice were killed on day 42, and sections of the hind paws were stained with hematoxylin and eosin (H&E) and immunostained with antibody against IP-10. B ⫽ bone; C ⫽ cartilage; BE ⫽ bone erosion; S ⫽ synovitis. B, Concentrations of IP-10 in sera from control mice and mice with CIA, as measured by enzyme-linked immunosorbent assay. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.01 versus controls, by one-way analysis of variance. All results are representative of 3 independent experiments. RESULTS Expression of IP-10 in inflamed joints of mice with CIA. To investigate whether IP-10 is expressed in the synovium in RA, we used a mouse model of RA. Histologic analysis of the paws showed that the inflamed joints of mice with CIA were massively infiltrated with mononuclear cells, and cartilage destruction and bone erosion of the joint were greater in CIA mice than in control mice. IP-10 was slightly expressed in the control mice; expression was greatly increased in the inflamed joints of CIA mice (Figure 1A). In addition, IP-10 expression in serum was significantly higher in CIA mice than in control mice (Figure 1B). We also found that IP-10 concentrations were greatly increased in serum and synovial fluids from RA patients as compared with osteoarthritis patients (data not shown). These results suggest that IP-10 is significantly elevated in the synovium in RA and likely contributes to the progression of CIA in mice. RANKL induction of IP-10 expression by osteoclast precursors. The induction of IP-10 in inflamed joints may play an important role in CIA, which raises the question of how the IP-10 levels are increased in inflamed joints. Levels of inflammatory cytokines such as TNF␣ and IL-1 have been shown to be significantly higher in the synovial tissue or synovial fluid in patients 1335 with RA (22). We examined whether TNF␣ and IL-1 regulate the expression of IP-10 in mouse synovial fibroblasts. TNF␣ and IL-1 did not induce IP-10 expression in synovial fibroblasts. However, TNF␣ and IL-1 induced the expression of IP-10 in cocultures of mouse synovial fibroblasts and osteoclast precursors. The induction of IP-10 in cocultures was decreased by the addition of OPG (data not shown). Therefore, to determine whether RANKL mediates the expression of IP-10 in osteoclast precursors, mouse cells were stimulated with RANKL for various times. RT-PCR analysis showed that the expression of IP-10 increased from 6 hours to 24 hours in response to RANKL (Figure 2A). M-CSF alone did not induce IP-10 expression and had no synergistic effect on IP-10 expression in response to RANKL (Figure 2B). Furthermore, RANKL directly induced IP-10 expression in osteoclast precursors, without new protein synthesis, but did not induce IP-10 expression in mature osteoclasts (Figures 2C and D). To confirm this result, levels of IP-10 protein in osteoclast precursors were measured by Western blotting. Consistent with the results of the RT-PCR analyses, RANKL induced the expression of IP-10 protein in a time-dependent manner (Figure 2E). By 24 hours after RANKL treatment, protein levels of IP-10 increased in a dose-dependent manner (Figure 2F). To determine the amount of IP-10 that was secreted, the level of IP-10 in the culture medium was determined by ELISA, and the results were consistent with those of the Western blot analysis (Figure 2G). These results suggest that RANKL mediates the induction of IP-10 in osteoclast precursors. IP-10 up-regulation of osteoclastogenic cytokine expression in CD4ⴙ T cells. To examine the hypothesis that the IP-10 expressed by osteoclast precursors plays a critical role in the activation of T cells in the synovium of inflamed joints, we first investigated whether IP-10 induces the expression of NF-AT because NF-AT induction is essential for T cell activation. Western blot analysis showed that IP-10 induces the expression of NF-ATc1 and NF-ATc2 and stimulates the phosphorylation of ERK, Akt, and p38 MAPK in CD4⫹ T cells (Figure 3A). Because it has been reported that activated T cells express RANKL and mediate osteoclastogenesis (23), we examined whether IP-10 induces the expression of RANKL or of mediators of inflammation in CD4⫹ T cells. As shown in Figure 3B, IP-10 significantly induced RANKL and TNF␣, but the levels of COX-2, IL-1␤, IL-17, and IFN␥ gradually decreased to basal levels or below. Cycloheximide had no appreciable effect on the 1336 KWAK ET AL Figure 2. RANKL induction of interferon-␥–inducible protein 10 (IP-10) expression in osteoclast precursors. A, Osteoclast precursors were stimulated with RANKL (100 ng/ml) in the presence of macrophage colony-stimulating factor (M-CSF; 30 ng/ml) for the indicated times. B, Osteoclast precursors were stimulated with M-CSF (30 ng/ml), RANKL (100 ng/ml), or both for 6 hours. C, Osteoclast precursors were pretreated or not pretreated with cycloheximide (CHX; 3 g/ml) for 30 minutes and further stimulated with RANKL (100 ng/ml) for the indicated times. D, Mature osteoclasts (MOC) and osteoclast precursors (M) were stimulated with RANKL (100 ng/ml) for the indicated times. In A–D, expression of mRNA for IP-10 was analyzed by reverse transcription–polymerase chain reaction. Values for each band were quantified by densitometry and were normalized to GAPDH intensity. E, Osteoclast precursors were stimulated as in A for the indicated times, and cell lysates (top) and culture media (middle) were analyzed by Western blotting with anti–IP-10 antibodies. The membrane was then stripped and reprobed with antibody to actin (bottom). F, Osteoclast precursors were stimulated with different concentrations of RANKL for 24 hours, and cell lysates (top) and culture media (middle) were analyzed by Western blotting as in E. The membrane was then stripped and reprobed with antibody to actin (bottom). G, Osteoclast precursors were stimulated as in F, and concentrations of IP-10 in the culture medium were measured by enzyme-linked immunosorbent assay. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.01 versus control (without RANKL), by one-way analysis of variance. All results are representative of at least 3 independent experiments. IP-10 MEDIATION OF BONE-EROSIVE EXPERIMENTAL ARTHRITIS 1337 Figure 3. Interferon-␥–inducible protein 10 (IP-10) activation of CD4⫹ T cells and induction of osteoclastogenic cytokines. A, Activated CD4⫹ T cells were obtained by positive selection using anti-CD4 magnetic beads and were stimulated with IP-10 (100 ng/ml) for the indicated times. Cell lysates were analyzed by Western blotting with antibodies to nuclear factor of activated T cells c1 (NF-ATc1), NF-ATc2, phospho-ERK, ERK, phospho-Akt, Akt, phospho-p38, p38, and actin. B, Activated CD4⫹ T cells were stimulated as in A, and the expression of mRNA for RANKL, tumor necrosis factor ␣ (TNF␣), cyclooxygenase 2 (COX-2), interleukin-1␤ (IL-1␤), IL-17, interferon-␥ (IFN␥), CXCR3, and GAPDH was analyzed by reverse transcription–polymerase chain reaction (RT-PCR). Values for each band were quantified by densitometry and were normalized to GAPDH intensity. C, Activated CD4⫹ T cells were pretreated or were not pretreated with cycloheximide (CHX; 3 g/ml) for 30 minutes and were further stimulated with IP-10 (100 ng/ml) for the indicated times. Expression of mRNA for the indicated genes was analyzed by RT-PCR. Densitometry values for each band were calculated as in B. D, Activated CD4⫹ T cells were stimulated with IP-10 (100 ng/ml) for the indicated times, and concentrations of RANKL, TNF␣, and IFN␥ in the culture media were analyzed by enzyme-linked immunosorbent assay. Values are the mean. E, CD8⫹ T cells were purified as in A and stimulated with IP-10 (100 ng/ml) for the indicated times. Expression of mRNA for the indicated genes was analyzed by RT-PCR. Densitometry values for each band were calculated as in B. All results are representative of at least 3 independent experiments. 1338 KWAK ET AL Figure 4. Enhanced osteoclast differentiation by interferon-␥–inducible protein 10 (IP-10) in cocultures of CD4⫹ cells and osteoclast precursors. A, Activated CD4⫹ T cells were cocultured with osteoclast precursors in the presence of the indicated concentrations of IP-10 and macrophage colony-stimulating factor (M-CSF; 10 ng/ml) for 6 days. Cells were fixed and stained with tartrate-resistant acid phosphatase (TRAP) (top). TRAP-positive cells were counted as osteoclasts (bottom) and are shown as the mean and SD. ⴱ ⫽ P ⬍ 0.01 versus control (without IP-10), by one-way analysis of variance (ANOVA). B, Activated CD4⫹ T cells and osteoclast precursors were cocultured for 6 days with IP-10 (200 ng/ml) or IP-10 plus osteoprotegerin (OPG; 500 ng/ml) in the presence of M-CSF (10 ng/ml). Cells were fixed and stained with TRAP (top). TRAP-positive cells were counted as osteoclasts (bottom) and are shown as the mean and SD. ⴱ ⫽ P ⬍ 0.01 for the indicated comparison, by one-way ANOVA. All results are representative of at least 5 independent experiments. induction of RANKL or TNF␣ by IP-10 (Figure 3C). To confirm this result, the amount of RANKL, TNF␣, and IFN␥ secreted into the culture medium was measured by ELISA, and the results were consistent with the results of the RT-PCR analyses (Figure 3D). In contrast with the results in CD4⫹ T cells, IP-10 did not induce RANKL or TNF␣ expression in CD8⫹ T cells (Figure 3E). These results suggest that IP-10 activates CD4⫹ T cells and specifically induces the expression of RANKL and TNF␣ in CD4⫹ T cells. IP-10 enhancement of osteoclast differentiation in cocultures of CD4ⴙ T cells and osteoclast precursors. To examine the potential of IP-10 in osteoclastogenesis, osteoclast precursors were cocultured with CD4⫹ T cells in the presence of IP-10. We found that IP-10 induced TRAP-positive osteoclasts in a dose-dependent manner (Figure 4A). However, when osteoclast precursors were cocultured with CD8⫹ T cells, IP-10 did not mediate osteoclast differentiation (data not shown). Osteoclast differentiation induced by IP-10 was suppressed in the presence of OPG (Figure 4B). These results suggest that IP-10 induces osteoclast differentiation by inducing RANKL expression in CD4⫹ T cells. Suppression of CIA progression by treatment with neutralizing anti–IP-10 antibody. To investigate the role of IP-10 during the progression of CIA, we injected DBA/1 mice intradermally with the type II collagen emulsion, and on day 14, a booster injection of type II collagen was given, as well as an intravenous injection of an equal volume of control IgG (200 g/ mouse) or anti–IP-10 antibody (200 g/mouse). After a 4-week period without treatment, paw swelling in the CIA mice was found to be significantly reduced by the administration of anti–IP-10 antibody. Analysis by microfocal computed tomography showed that there was much less bone erosion in the mice injected with anti–IP-10 antibody than in the IgG-injected control mice (Figures 5A and B). Hematoxylin and eosin staining showed that the anti–IP-10 antibody significantly attenuated bone loss in the mice with CIA (Figure 5C). Importantly, CD4⫹ T cells and F4/80⫹ macrophages infiltrated into the synovium of mice with CIA and mice with CIA that had been injected with IgG, but anti– IP-10 antibody treatment inhibited the infiltration of CD4⫹ T cells and F4/80⫹ macrophages (Figures 5D and E). Furthermore, serum concentrations of RANKL IP-10 MEDIATION OF BONE-EROSIVE EXPERIMENTAL ARTHRITIS Figure 5. Amelioration of the progression of collagen-induced arthritis (CIA) by treatment with neutralizing antibody to interferon-␥–inducible protein 10 (IP-10). A, Photographs (top) and microfocal computed tomography (micro-CT) scans (bottom) of control (CTL) mice, mice with CIA, mice with CIA treated with IgG, and mice with CIA treated with anti–IP-10 antibody. On day 14 after the first immunization, mice were given a booster injection with an emulsion of type II collagen as well as an intravenous injection with an equal volume of control IgG (200 g per mouse; n ⫽ 5) or anti–IP-10 antibody (200 g per mouse; n ⫽ 5). On day 42, paws were photographed with a digital camera and 3-dimensional images were obtained with a micro-CT apparatus. Bone erosion values (right) were determined with a SkyScan CT analyzer. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05 for the indicated comparison, by one-way analysis of variance (ANOVA). B, Severity of arthritis, as assessed by the degree of swelling in each paw (scored 0–3) (see Materials and Methods for details). Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05 for the indicated comparisons, by one-way ANOVA. C, Histologic features of paws from the 4 groups of mice. Sections were stained with hematoxylin and eosin. B ⫽ bone; C ⫽ cartilage; BE ⫽ bone erosion; S ⫽ synovitis. D, Immunostaining of paws from the 4 groups of mice. Sections were immunostained with anti-CD4 antibody (left) and with F4/80 (right) as described in Materials and Methods. E, Number of immunostained cells in the 4 groups of mice. Cells immunostained with anti-CD4 or anti-F4/80 were quantified with the Image Pro-Plus program, version 4.0. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05 for the indicated comparisons, by one-way ANOVA. F, Serum levels of RANKL and tumor necrosis factor ␣ (TNF␣) in the 4 groups of mice, as determined by enzyme-linked immunosorbent assay. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05 for the indicated comparisons, by one-way ANOVA. All results are representative of 2 independent experiments. 1339 1340 KWAK ET AL retrovirus containing IP-10 to the mice. Retrovirus infection of cells was determined by confocal microscopy, and the infection efficiency of both retroviruses (pMXIP-10-IRES-EGFP and pMX-IRES-EGFP control) was ⬎90%. IP-10 protein was greatly increased in the IP-10 retrovirus–infected cells (Figure 6A). Next, control and IP-10 retroviruses were locally injected into the tibial metaphysis of each mouse. Bone erosion was greatly increased in IP-10 retrovirus–infected mice (Figure 6B). IP-10 is known to play a critical role in the recruitment of T cells; therefore, we also determined whether IP-10 induces the migration of CD4⫹ T cells and F4/80⫹ macrophages. IP-10 induced the migration of CD4⫹ T cells and osteoclast precursors in vitro (Figure 6C). These results show that IP-10 mediates bone erosion, which is associated with the migration of CD4⫹ T cells and F4/80⫹ macrophages to sites of inflammation. DISCUSSION Figure 6. Promotion of in vivo bone erosion by interferon-␥– inducible protein 10 (IP-10). Plat E cells were transiently transfected with pMX-IRES-EGFP (pMX; control) or pMX-IP-10-IRES-EGFP (pMX-IP-10) DNA to obtain retroviruses. Media containing retroviruses were collected 48 hours after transfection. A, Photomicrographs (left) and Western blots (right) of osteoclast precursors infected with control or IP-10 retrovirus in the presence of macrophage colonystimulating factor (30 ng/ml). Cells were infected for 48 hours, fixed, mounted, and examined by confocal microscopy or Western blotting with anti–IP-10 antibody. B, Radiographs of the knee joints of a control mouse, a mouse injected with control retrovirus, and a mouse injected with IP-10 retrovirus. Tibial metaphyses were injected with control (50 l; n ⫽ 5) or IP-10 (50 l; n ⫽ 5) retrovirus once a week for 6 weeks. Mice were then killed, and radiographs were obtained with a soft x-ray machine. Arrows indicate bone erosions. C, Migration of CD4⫹ T cells (left) and osteoclast precursors (right) in response to IP-10. Cell migration was analyzed by using a Boyden chamber. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05 versus control (CTL), by one-way analysis of variance. All results are representative of at least 3 independent experiments. and TNF␣ were significantly lower in mice with CIA that had been injected with anti–IP-10 antibody than in mice with CIA or mice with CIA injected with IgG (Figure 5F). These results suggest that the induction of IP-10 in the synovium of inflamed joints is important for the progression of RA. IP-10 promotion of bone erosion through recruitment of CD4ⴙ T cells and macrophages. To examine whether IP-10 can induce the infiltration of inflammatory cells, such as F4/80⫹ macrophages and CD4⫹ T cells, and cause bone erosion in vivo, we administered a Inflammatory cytokines produced by macrophages and lymphocytes at sites of inflammation play an important role in bone-related diseases such as RA (4,12). The regulation of osteoclast formation induced by inflammatory cytokines is vital for preventing inflammation-induced bone loss. Although the mechanism of osteoclast differentiation is well known, how osteoclast precursors or osteoclasts are recruited into bone tissue is not well known. Using our mouse model of CIA, we found that IP-10 is increased in inflamed joints (Figure 1), which suggests that IP-10 plays an important role in the progression of RA. Postmenopausal osteoporosis is a predominant bone disease that is caused by increased bone resorption resulting from a complete deficiency of estrogen rather than inflammation. Thus, we examined serum concentrations of IP-10 in osteoporosis patients, but found that these were not elevated compared with concentrations in healthy humans (data not shown). These results show that the up-regulation of IP-10 is involved in bone diseases that are mediated by inflammation, such as RA. IP-10 is expressed in the synovium in RA and is increased in vitro by the coculture of synoviocytes and monocytes. However, the induction of IP-10 in monocytes is not reduced by the addition of antibodies against IFN␥ or TNF␣ (21). Although many studies have shown that IP-10 is mainly secreted by monocytes and macrophages in response to IFN␥, lipopolysaccharide, and TNF␣, IFN␥ is not expressed in the synovial tissue or synovial fluid in RA (24) and has a robust inhibitory effect on osteoclast differentiation (25). Therefore, it is IP-10 MEDIATION OF BONE-EROSIVE EXPERIMENTAL ARTHRITIS hard to define exactly how IP-10 is induced in the synovium in RA. It is well known that IFN␥ mediates the expression of monokine induced by IFN␥ (MIG) in various cell types. Although MIG is expressed in IFN␥-treated cells, we previously showed that MIG is induced by RANKL, which in turn, induces the migration of osteoclast precursors and osteoclasts (21). MIG was also shown to be increased in the synovial fluid of RA patients (26). RANKL is expressed in synovial tissue, synovial fibroblasts, chondrocytes, and mesenchymal cells in the cartilage as well as in osteoblasts and activated T cells (27). Therefore, we examined the ability of RANKL to regulate the expression of IP-10. RANKL significantly induced IP-10 expression in osteoclast precursors (Figure 2). These results strongly suggest that RANKL is associated with IP-10 secretion in the synovium of inflamed joints. The accumulation of monocytes, macrophages, and lymphocytes in the synovium in response to various chemokines is important in the initiation of RA. Inflammatory cytokines such as IL-1, TNF␣, and IL-6 are important mediators of inflammation that have been shown to be involved in the pathogenesis of RA (28). In the context of RA, bone-resorbing osteoclasts are differentiated from osteoclast precursors and immature osteoclasts exposed to inflammatory cytokines such as RANKL, TNF␣, IL-1, and IL-17, which are expressed by activated T cells (CD4⫹) and by macrophages in the inflamed joint (22). In particular, TNF␣ is a multifunctional cytokine that is mainly expressed by activated macrophages; it causes the production of other cytokines, including IL-1, IL-6, and RANKL. The mouse model of CIA that we used has pathologic features similar to those in humans with RA. Enhanced levels of TNF␣ play a critical role in inflammation and mediate cartilage and bone destruction in CIA (29). Consistent with previous reports, we found that serum concentrations of TNF␣ and RANKL were greatly elevated in mice with CIA, but the up-regulation of TNF␣ and RANKL was inhibited by neutralizing antibody to IP-10, which also suppressed the infiltration of F4/80⫹ macrophages and CD4⫹ T cells into inflamed joints (Figure 5). We also found that IP-10 mediates the migration of F4/80⫹ macrophages and CD4⫹ T cells (Figure 6). Our results strongly suggest a critical role of IP-10 in the infiltration of F4/80⫹ macrophages and CD4⫹ T cells into the synovium and in the regulation of TNF␣ and RANKL. Previous studies have shown that synovial T cells express RANKL and that RANKL is only expressed in T 1341 cells in the synovium of RA patients and not in synovial fibroblasts (30,31). However, other studies have shown that RANKL is expressed in synovial fibroblasts and synovial tissue, but at lower levels than in T cells (12). Thus, we examined the effect of IP-10 in RANKL or TNF␣ expression in T cells as well as in synovial fibroblasts and macrophages, which are resident in the synovium and play an important role in inflammation in the inflamed joints. We found that IP-10 induced the expression of RANKL and TNF␣ in CD4⫹ T cells but not in synovial fibroblasts, macrophages, or CD8⫹ T cells (Figure 3 and data not shown). These results suggest that IP-10 specifically activates CD4⫹ T cells and promotes the increased expression of RANKL and TNF␣ in the inflamed joints. Induction of RANKL in activated T cells has been shown to be regulated by protein kinase C, phosphatidylinositol 3-kinase, ERK, and calcineurin signaling pathways (12). We found that IP-10 stimulates the sustained activation of ERK and Akt in CD4⫹ T cells (Figure 3), which implies that these signaling pathways might be involved in the IP-10 induction of RANKL and TNF␣ in CD4⫹ T cells. We showed that IP-10 significantly induces osteoclast differentiation in a coculture of osteoclast precursors and CD4⫹ T cells by inducing RANKL in CD4⫹ T cells (Figure 4). These results suggest that IP-10–induced production of RANKL in synovial T cells plays a critical role in osteoclast formation in RA. In addition, similar to CD40 ligand, T cell–derived RANKL in the immune system is important for promoting CD4⫹ T cell proliferation and mediates the survival of dendritic cells through interaction with RANK (4). We provide strong evidence that RANKL promotes the expression of IP-10 in osteoclast precursors and that IP-10 mediates RANKL expression in T cells (CD4⫹) in the synovium. The reciprocal cross-talk between RANKL and IP-10 is responsible for inflammation and bone erosion by recruiting CD4⫹ T cells and macrophages into the synovium. IP-10 might be one of the therapeutic targets in RA, and blockade of IP-10 may be an important therapeutic target in the prevention of bone destruction in RA. AUTHOR CONTRIBUTIONS Dr. Z. H. Lee had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design. S. Lee, H.-H. Kim, Z. H. Lee. Acquisition of data. Kwak, H.-N. Kim, J.-H. Lee, H.-M. Kim, J. Y. Kim, Song. Analysis and interpretation of data. Kwak, Ha, Song, Z. H. Lee. Manuscript preparation. Kwak, Z. H. Lee. Statistical analysis. H. S. Kim. 1342 KWAK ET AL REFERENCES 1. Teitelbaum SL, Ross FP. Genetic regulation of osteoclast development and function. Nat Rev Genet 2003;4:638–49. 2. Krane SM. Identifying genes that regulate bone remodeling as potential therapeutic targets. J Exp Med 2005;201:841–3. 3. Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science 2000;289:1508–14. 4. Theill LE, Boyle WJ, Penninger JM. 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