Effect of IL-12 on TNF-α-Mediated Osteoclast Formation in Bone Marrow Cells: Apoptosis Mediated by Fas/Fas Ligand Interaction This information is current as of October 6, 2021. Hideki Kitaura, Noriko Nagata, Yuji Fujimura, Hitoshi Hotokezaka, Noriaki Yoshida and Koji Nakayama J Immunol 2002; 169:4732-4738; ; doi: 10.4049/jimmunol.169.9.4732 http://www.jimmunol.org/content/169/9/4732 Downloaded from References This article cites 54 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/169/9/4732.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 6, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Effect of IL-12 on TNF-␣-Mediated Osteoclast Formation in Bone Marrow Cells: Apoptosis Mediated by Fas/Fas Ligand Interaction1 Hideki Kitaura,2* Noriko Nagata,*† Yuji Fujimura,*† Hitoshi Hotokezaka,* Noriaki Yoshida,* and Koji Nakayama† Recently, it has been found that differentiation into osteoclasts is induced by TNF-␣. In this study, we investigated the effect of IL-12 on TNF-␣-mediated osteoclastogenesis. When mouse bone marrow cells were cultured with TNF-␣, osteoclast-like cells were formed. When they were cultured with both TNF-␣ and IL-12, the number of adherent cells in the bone marrow cells decreased in an IL-12 dose-dependent manner. A combination of IL-12 and TNF-␣ was necessary to induce death of the adherent cells in this culture system. Apoptotic alterations, which were indicated by morphological changes such as cellular atrophy, nuclear and cellular fragmentation, and biochemical changes such as DNA fragmentation, were observed in the adherent cells. Apoptosis of Downloaded from the adherent cells was markedly inhibited by anti-Fas ligand (FasL) Ab. RT-PCR and FACS analyses revealed that TNF-␣ up-regulated Fas transcription to lead to Fas expression on the surfaces of the adherent cells, whereas IL-12 could not induce Fas on the cells. In contrast, IL-12 induced FasL transcription to lead to FasL expression on the surfaces of nonadherent bone marrow cells, whereas TNF-␣ could not induce FasL on the cells. These results implied that apoptosis of the adherent cells in bone marrow cells might be caused by interaction between TNF-␣-induced Fas on the adherent cells and IL-12-induced FasL on the nonad- herent cells. The Journal of Immunology, 2002, 169: 4732–4738. http://www.jimmunol.org/ one resorption is controlled by osteoclasts that are dif- Kobayashi et al. (12) have reported that TNF-␣ induces oste- ferentiated from hematopoietic stem cells (1–3). Bone oclast-like cells from M-CSF-dependent bone marrow-derived B marrow contains only a small number of mature oste- macrophages in vitro. TNF-␣ is known to play a major role in host oclasts in a nonproliferative state because mature osteoclasts are defense, and it exerts proinflammatory activities through various multinuclear giant cells formed by fusion of mononuclear cells. cells including mononuclear phagocytes, in which it is responsible Formation of mature osteoclasts requires two cellular factors: for the activation of bactericidal/cytocidal systems (13, 14). ␬ 3 M-CSF and the receptor activator of NF- B ligand (RANKL). TNF-␣ is involved in differentiation into both osteoclasts and mac- by guest on October 6, 2021 M-CSF is indispensable for proliferation and/or differentiation of rophages, although their biological roles seem quite different. osteoclast precursors (4, 5). The osteopetrotic op/op mice are ex- TNF-␣ is pleiotropic, which has a variety of often opposing tremely deficient in osteoclasts and macrophages. This deficiency biological effects in a cell-specific manner. As one of its most is caused by the absence of functional M-CSF and can be cured by perplexing properties, TNF-␣ promotes cell survival in certain injections of M-CSF (6). In contrast, RANKL has been identified conditions and cell death in others (15). The findings that TNF-␣ as a ligand for RANK, an immunoresponsive receptor on dendritic recognizes two receptors on cell surfaces (type 1 or p55 and type cells (7). RANKL, which is also called TNF-related activation- 2 or p75 receptors) and that each receptor is capable of distinct induced cytokine (8), osteoclast differentiation factor (9), and os- intracellular signaling (15) have substantially deepened under- teoprotegerin ligand (10), can cause differentiation from osteoclast standing of the complex activities of the cytokine. TNF-␣-induced precursors into mature osteoclasts in the presence of M-CSF in in osteoclast recruitment is probably central to the pathogenesis of vitro culture systems. RANKL-deficient mice show severe osteo- disorders with inflammatory osteolysis such as periodontal disease petrosis and lack osteoclasts completely (11). (16) and periprosthetic bone loss (17). In fact, TNF-␣ is shown to be involved in the causes of postmenopausal osteoporosis (18, 19). *Divisions of Orthodontic and Biomedical Engineering and †Microbiology and Oral IL-12 is a heterodimeric 70-kDa protein that is composed of two Infection, Department of Developmental and Reconstructive Medicine, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical subunits (p35 and p40) linked by a disulfide bond. IL-12 has been Sciences, Nagasaki, Japan found to have the ability to induce maturation of cytotoxic T lym- Received for publication June 20, 2002. Accepted for publication August 22, 2002. phocytes and to enhance production of IFN-␥ in NK cells (20–22). The costs of publication of this article were defrayed in part by the payment of page IL-12 has also been shown to have a pivotal role in Th1-dominant charges. This article must therefore be hereby marked advertisement in accordance immune responses such as host defense responses against intra- with 18 U.S.C. Section 1734 solely to indicate this fact. cellular pathogens (23–25). 1 This work was supported in part by a Grant-in-Aid (13771268) for Scientific Re- search from the Ministry of Education, Science, Sports and Culture, Japan. In a recent study, mouse and human osteoblasts in vitro infected with Staphylococcus aureus have been found to express high lev- 2 Address correspondence and reprint requests to Dr. Hideki Kitaura, Divisions of Orthodontic and Biomedical Engineering, Department of Developmental and Recon- els of IL-12 (26). More recently, Horwood and colleagues (27, 28) structive Medicine, Course of Medical and Dental Sciences, Nagasaki University have found that IL-12 can inhibit osteoclast formation in spleen Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Ja- pan. E-mail address: [email protected] cell cultures in vitro and that the IL-12-mediated inhibition of 3 Abbreviations used in this paper: RANKL, receptor activator of NF-␬B ligand; osteoclast formation is T cell dependent, like the inhibitory ac- FasL, Fas ligand; TRAP, tartrate-resistant acid phosphatase. tion of IL-18. Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 The Journal of Immunology 4733 ␣ In this study, we investigated the effect of IL-12 on TNF- - 3 mM MgCl2, 0.1% Triton X-100, 10 mM DTT, 0.5 mM deoxynucleotide induced osteoclastogenesis in bone marrow cells. triphosphates, 5 ␮M random hexamer primers (Promega, Madison, WI), and 40 U/␮l Moloney murine leukemia virus reverse transcriptase (Invitro- gen, Carlsbad, CA)) and were incubated at 42°C for 50 min followed by Materials and Methods incubation at 70°C for 15 min. The resulting cDNA was then diluted to 100 Animals and reagents ␮l with distilled water. PCR amplification was performed in a reaction mixture (50 ␮l) containing the cDNA solution (5 ␮l), 10 mM Tris ⅐ HCl Five-week-old male ddY mice were purchased from Seac Yoshitomi (pH 8.3), 50 mM KCl, 2.5 mM MgCl , 0.1% Triton X-100, 0.2 mM de- (Fukuoka, Japan). Recombinant human M-CSF was purchased from Yo- 2 ␣ oxynucleotide triphosphates,1UofTaq polymerase (Wako Nippon Gene, shitomi Pharmaceutical (Tokyo, Japan) and recombinant mouse TNF- Tokyo, Japan), and 1 mM appropriate primers. The primers used were as was purchased from R&D Systems (Minneapolis, MN). Recombinant follows: sense, 5Ј-TCCTGTGGCATCCATGAAACT-3Ј, and antisense, mouse IL-12 was obtained from Wako Pure Chemical (Osaka, Japan), and 5Ј-CTTCGTGAACGCCACGTGCTA-3Ј, for ␤-actin; sense, 5Ј-TTGCT anti-Fas and anti-Fas ligand (FasL) Abs were from BD PharMingen (San GTCAACCATGCCAAC-3Ј, and antisense, 5Ј-ACGTGAACCATAAGAC Diego, CA). CCAG-3Ј, for Fas; sense, 5Ј-ATCCCTCTGGAATGGGAAGA-3Ј, and an- Ј Ј TNF-␣-induced osteoclast formation in bone marrow cells tisense, 5 -CCATATCTGTCCAGTAGTGC-3 , for FasL. The conditions for amplification were as follows: one cycle (93°C, 3 min), 30 cycles The femora and tibiae of mice were aseptically removed and dissected free (93°C, 1 min; 55°C, 1 min; 72°C, 2 min), and one cycle (72°C, 7 min) for of adhering tissues. The bone ends were cut off by scissors and the marrow ␤-actin and FasL; and one cycle (93°C, 3 min), 30 cycles (93°C, 1 min; cavity was flushed out by slow injection of ␣-MEM (Sigma-Aldrich, To- 63°C, 1 min; 72°C, 2 min), and one cycle (72°C, 7 min) for Fas.