Interaction Apoptosis Mediated by Fas/Fas Ligand Osteoclast Formation In

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

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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 © 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 nonadherent 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.

␣ 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. kyo, Japan) at one end of the bone using a sterile needle to collect bone marrow cells. After washing with ␣-MEM, cells were incubated in culture FACS and flow cytometry medium (␣-MEM containing 10% FBS, 100 IU/ml penicillin G (Meiji The adherent and nonadherent cells of bone marrow cells were incubated Seika, Tokyo, Japan), and 100 ␮g/ml streptomycin (Meiji Seika)). Whole for 15 min with the monoclonal mouse Abs raised against Fas and FasL. Downloaded from bone marrow cells were cultured at 2 ϫ 106 cells/ml in a 48-well plate in After being washed with PBS, cells were incubated with FITC-conjugated the presence of M-CSF (50 ng/ml) and TNF-␣ (50 ng/ml) for 1–5 days donkey anti-mouse Ab (Sigma-Aldrich) for 30 min, washed, diluted with without medium change in the cultures. M-CSF, TNF-␣, and IL-12 were 20 ml of PBS, and subjected to FACS analysis. Samples were analyzed used at a final concentration of 50 ng/ml in this study, except in the ex- using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ) for periments with indicated concentrations. detection of Fas and FasL. Tartrate-resistant acid phosphatase (TRAP) staining Statistical analysis Cultured cells were fixed with 4% paraformaldehyde for 30 min and then http://www.jimmunol.org/ Differences between data were analyzed with the Student t test. 0.2% Triton X-100 for 5 min at room temperature and were incubated in acetate buffer (pH 5.0) containing naphthol AS-MX phosphate (Sigma- Aldrich), fast red-violet LB salt (Sigma-Aldrich), and 50 mM sodium Results tartrate. In vitro osteoclast differentiation by TNF-␣ Bone marrow-derived macrophages Initially, we examined whether TNF-␣ could induce osteoclast differentiation in an in vitro culture system of bone marrow cells. Bone marrow cells were incubated in culture medium supplemented with M-CSF (100 ng/ml) at 1 ϫ 107 cells per 10 ml in a 10-cm culture dish. TRAP-positive cells were found after culturing bone marrow cells After 3-day culture, cells were washed vigorously with PBS twice to re- in the presence of 100 ng/ml TNF-␣ for 3 days, although no move nonadherent cells, harvested by pipetting with 0.02% EDTA in PBS, TRAP-positive cells were detected in the cultures with TNF-␣ of by guest on October 6, 2021 ϫ 6 and seeded at 1 10 cells per 10 ml in a 10-cm dish. After an additional concentrations lower than 1 ng/ml or in the culture in the presence 3-day culture, cells were harvested. We used these cells as bone marrow macrophages in this study (29). of M-CSF alone (Figs. 1 and 2). The number of TRAP-positive cells increased along with increase of TNF-␣ (10–100 ng/ml) in 4- Cell viability assay and 5-day cultures (Fig. 1). Most of the TRAP-positive cells were Cell viability was determined by the MTT assay. Cultures were washed mononucleated until day 5. When M-CSF and TNF-␣ were added with PBS twice to remove nonadherent cells. The adherent cells were cul- again to the cultures at day 5, multinucleated cells appeared in tured in 1 ml of culture medium in a well. Ten microliters of MTT (10 7-day culture (data not shown). mg/ml) were added to each well, and the mixture was incubated for4hat 37°C. SDS was then added to the mixture at 10% and incubated for 3 h. Culture medium was then replaced with DMSO to dissolve formazan crys- tals. After shaking at room temperature for 10 min, absorbance of each well was determined at 570 nm using a microplate reader (model 550; Bio-Rad, Richmond, CA). Samples were measured in three replicates and each experiment was repeated at least twice. DNA fragmentation DNA was isolated from eukaryotic cells as previously described (30) and was subjected to electrophoresis on a 1.5% agarose gel containing ethidium bromide. DNA was visualized under UV light. Nuclear morphology Cultures in a chamber slide system (Nalge Nunc International, Naperville, IL) were washed twice with PBS to remove nonadherent cells and were fixed with 4% formaldehyde in PBS for 30 min at room temperature. After washing with PBS twice, cells were stained with Hoechst 33342 (Sigma- Aldrich) (working dilution, 1/1000) at 37°C for 30 min and were observed by fluorescence microscopy. FIGURE 1. Effect of TNF-␣ on formation of TRAP-positive cells in RNA preparation and analysis bone marrow cells. Bone marrow cells of ddY mice were cultured with 50 ␣ Total RNA was isolated from adherent cells and nonadherent cells that had ng/ml M-CSF and various concentrations of TNF- for 1–5 days. Cells been cultured for 72 h using TRIzol reagent (Life Technologies, Grand were washed to remove nonadherent cells and were then fixed and stained Island, NY). For RT-PCR analysis, 2 ␮g of RNA samples were dissolved for TRAP. The number of TRAP-positive cells was scored. Results were in 20 ␮l of the reaction mixture (50 mM Tris ⅐ HCl (pH 8.3), 75 mM KCl, expressed as mean Ϯ SEM of three cultures. 4734 IL-12 AND TNF-␣ INDUCE APOPTOSIS IN BONE MARROW CELL CULTURES

TNF-␣, the number of TRAP-positive cells was markedly decreased in a dose-dependent fashion (Figs. 2 and 3). The number of adherent cells was also decreased and the remaining adherent cells showed cytopathic changes (Fig. 2C). Apoptotic alterations such as atrophy and cytoclasis were observed in these cells. No TRAP-positive cells were formed and no cytopathic changes were observed when both M-CSF and IL-12 were added to culture medium in the absence of TNF-␣ (Fig. 2D). We examined these cells for viability using MTT assay. When bone marrow cells were cultured in the presence of M-CSF and TNF-␣, viability of these cells was slightly decreased, compared with that in the presence of M-CSF alone. When bone marrow cells were cultured in the presence of M-CSF, TNF-␣, and IL-12, viability of the cells was decreased to A). Decrease of viability was dependent on the concentration of IL-12 (Fig. 4B). Cell viability after culturing in the presence of both M-CSF and IL-12 was similar to that after culturing in the presence of M-CSF alone (Fig. 4A). These results indicated that death of bone marrow cells was induced by culturing with a combination of M-CSF, TNF-␣, and IL-12. Downloaded from Nuclear fragmentation The morphology of nuclei of the adherent cells was investigated using the nuclear stain Hoechst 33342. The adherent cells in the http://www.jimmunol.org/

FIGURE 2. Effect of IL-12 on TNF-␣-induced formation of TRAP-positive cells in bone marrow cells. Bone marrow cells were treated with M-CSF (A); M-CSF and TNF-␣ (B); M-CSF, TNF-␣, and IL-12 (C); and M-CSF and IL-12 (D) for 4 days. Cells were washed to remove nonadherent cells and were then ﬁxed and stained for TRAP.

Effect of IL-12 on TNF-␣-induced osteoclast formation from by guest on October 6, 2021 bone marrow cells To examine the effect of IL-12 on TNF-␣-induced osteoclast formation from bone marrow cells, various concentrations of IL-12 were added to the culture of bone marrow cells with M-CSF and TNF-␣. When IL-12 was added to the culture with M-CSF and

FIGURE 4. Effect of IL-12 on viability of the adherent cells in bone marrow cells. A, Time course. Bone marrow cells were treated with M-CSF alone (lane 1); M-CSF and TNF-␣ (lane 2); M-CSF, TNF-␣, and IL-12 (lane 3); and M-CSF and IL-12 (lane 4) for 1–4 days. Viability was determined by the MTT assay. Samples were measured in three replicates and each experiment was repeated at least twice. Data are presented as a percentage of relative activity against the activity obtained from the culture p Ͻ ,ءء ;p Ͻ 0.05 ,ء .with M-CSF alone and expressed as mean Ϯ SEM FIGURE 3. Effect of IL-12 on the number of TRAP-positive cells in 0.01; related to the activity of the culture in the presence of M-CSF. B, bone marrow cells in the presence of M-CSF and TNF-␣. Bone marrow IL-12 concentration. Bone marrow cells were treated with M-CSF (50 ng/ cells of ddY mice were cultured with M-CSF (50 ng/ml), TNF-␣ (50 ng/ ml), TNF-␣ (50 ng/ml), and an indicated concentration of IL-12 for 4 days. ml), and an indicated concentration of IL-12 for 4 days. After washing with Viability was determined by the MTT assay. Samples were measured in PBS to remove nonadherent cells, cells were ﬁxed and the number of three replicates and each experiment was repeated at least twice. Data are TRAP-positive cells was scored. Results were expressed as mean Ϯ SEM presented as a percentage of relative activity against the activity obtained ;p Ͻ 0.05 ,ء .p Ͻ 0.01; related to the activity of the from the culture without IL-12 and expressed as mean Ϯ SEM ,ءء ;p Ͻ 0.05 ,ء .of three cultures .p Ͻ 0.01; related to the activity of the culture without IL-12 ,ءء .culture without IL-12 The Journal of Immunology 4735 culture with M-CSF alone, M-CSF and TNF-␣, and M-CSF and IL-12 showed round nuclei. In contrast, nuclear fragmentation was observed in the adherent cells cultured with a combination of M-CSF, TNF-␣, and IL-12 (Fig. 5).

DNA fragmentation Fig. 6 shows that DNA fragmentation resulting in DNA ladders was observed in the adherent cells in the culture with a combination of M-CSF, TNF-␣, and IL-12. No DNA fragmentation was observed in the adherent cells when bone marrow cells were cultured in the presence of M-CSF alone, M-CSF and TNF-␣,or FIGURE 6. Fragmentation of the chromosomal DNA of the adherent ␣ M-CSF and IL-12. cells treated with M-CSF, TNF- , and IL-12. Bone marrow cells were treated with M-CSF (lane 1); M-CSF and TNF-␣ (lane 2); M-CSF, TNF-␣, No effect of IL-12 on TNF-␣-induced osteoclast formation from and IL-12 (lane 3); and M-CSF and IL-12 (lane 4) for 4 days. M-CSF, ␣ bone marrow macrophages TNF- , and IL-12 were used at 50 ng/ml. Cells were washed with PBS to remove nonadherent cells. Chromosomal DNA was isolated from the ad- Because whole bone marrow cells consisted of various types of herent cells and subjected to electrophoresis on a 1.5% agarose gel con- cells, it remained unclear whether IL-12 directly acted on precur- taining ethidium bromide. DNA was visualized under UV light. MW, m.w. sors of osteoclasts. Therefore, direct actions of IL-12 on bone mar- standard, ␭DNA-HindIII digest. row macrophages enriched by treatment with M-CSF as osteoclast Downloaded from precursor cells were determined in TNF-␣-induced osteoclast formation. As shown in Fig. 7, when bone marrow macrophages were Induction of apoptosis of bone marrow cells in the presence of ␣ treated with M-CSF and TNF- , a number of TRAP-positive cells M-CSF, TNF-␣, and IL-12 by Fas/FasL interaction were formed. In contrast to the results of whole bone marrow cells, TRAP-positive cells were equally formed in the cultures of bone Fas/FasL interaction has been reported to induce apoptosis (31). marrow macrophages treated with M-CSF and TNF-␣ in the pres- Therefore, we investigated whether the induction of apoptosis of ence or absence of IL-12. Apoptotic alterations such as atrophy the adherent cells in bone marrow cells could be caused by Fas/ http://www.jimmunol.org/ and cytoclasis were not observed in bone marrow macrophages. FasL interaction. Apoptotic changes of bone marrow cells treated ␣ These results indicated that apoptosis might not be induced by a with a combination of M-CSF, TNF- , and IL-12 were markedly direct action of IL-12 on precursors of osteoclasts, but by some inhibited by addition of anti-FasL to the culture (Fig. 8A). The factors induced by an action of IL-12 on nonosteoclast precursor decrease of viability of the adherent cells in bone marrow cells ␣ cells in bone marrow cells. treated with a combination of M-CSF, TNF- , and IL-12 was also inhibited by addition of anti-FasL; however, cell viability in the culture with anti-FasL did not reach the level of cell viability in the culture without TNF-␣ or IL-12 (Fig. 8B). Also, the addition of by guest on October 6, 2021 anti-FasL Abs markedly inhibited the decrease of the number of TRAP-positive cells by treatment with M-CSF, TNF-␣, and IL-12 (Fig. 8C). Although the number of TRAP-positive cells in the culture with M-CSF, TNF-␣, IL-12, and anti-FasL did not reach the level of that in the culture with M-CSF and TNF-␣, the rate of anti-FasL-mediated recovery in the number of TRAP-positive cells

FIGURE 5. Nuclear morphology of the adherent cells in bone marrow FIGURE 7. Effect of IL-12 on TNF-␣-induced osteoclast formation cells. Bone marrow cells were treated with M-CSF (A); M-CSF and TNF-␣ from bone marrow macrophages. Bone marrow macrophages were treated (B); M-CSF, TNF-␣, and IL-12 (C); and M-CSF and IL-12 (D) for 4 days. with M-CSF (A), M-CSF and TNF-␣ (B), M-CSF and IL-12 (D), and M-CSF, TNF-␣, and IL-12 were used at 50 ng/ml. After being washed with M-CSF, TNF-␣, and IL-12 (E) for 3 days; and M-CSF and IL-12 (C) and PBS to remove nonadherent cells, the resulting adherent cells were ﬁxed M-CSF, TNF-␣, and IL-12 (F) for 5 days. M-CSF, TNF-␣, and IL-12 were and stained with Hoechst 33342. used at 50 ng/ml. Cells were ﬁxed and stained for TRAP. 4736 IL-12 AND TNF-␣ INDUCE APOPTOSIS IN BONE MARROW CELL CULTURES

Effect of IL-12 on Fas and FasL expression Adherent cells in the cultures with M-CSF alone and M-CSF and IL-12 showed no Fas mRNA, whereas Fas mRNA was clearly detected in the adherent cells in the culture with M-CSF and TNF-␣. Nonadherent cells in the cultures with M-CSF alone and M-CSF and TNF-␣ showed no FasL mRNA, whereas FasL mRNA was clearly detected in the nonadherent cells in the culture with M-CSF and IL-12 (Fig. 9A). FACS analysis revealed that TNF-␣ induced Fas on the surfaces of the adherent cells, whereas IL-12 failed to induce Fas on the cell surfaces. In contrast, IL-12 induced FasL on the surfaces of the nonadherent cells, whereas TNF-␣ could not induce FasL on the cell surfaces (Fig. 9B). Therefore, both IL-12 and TNF-␣ were necessary to induce apoptosis in this culture system. The results strongly suggested that apoptosis of the adherent cells in the bone marrow cell cultures might be caused by interaction between TNF- ␣-induced Fas on the adherent cells and IL-12-induced FasL on the nonadherent cells. Downloaded from Discussion It has been believed that RANKL is the essential factor responsible for osteoclast differentiation since the discovery of the RANK- RANKL signal transduction. This notion is supported by the ﬁnd- ing that the targeted disruption of the gene encoding RANKL in

mice develops severe osteopetrosis with the complete absence of http://www.jimmunol.org/ TRAP-positive cells in bone tissues (11). However, Kobayashi et al. (12) have recently found that TNF-␣ together with M-CSF induces formation of TRAP-positive osteoclast-like cells in bone by guest on October 6, 2021

FIGURE 8. Inhibitory effect of anti-FasL Ab on IL-12/TNF-␣-induced apoptosis of bone marrow cells. A, Microscopic observation. Bone marrow cells were treated with M-CSF (a); M-CSF and TNF-␣ (b); M-CSF, TNF-␣, and IL-12 (c); and M-CSF, TNF-␣, IL-12, and anti-FasL Ab (1 ␮g/ml) (d) for 4 days. Cells were washed to remove nonadherent cells, fixed, and stained for TRAP. B, MTT assay. Bone marrow cells were treated with M-CSF (lane 1); M-CSF and TNF-␣ (lane 2); M-CSF, TNF-␣, and IL-12 (lane 3); and M-CSF and IL-12 (lane 4) with or without anti- FasL Ab (1 ␮g/ml) for 4 days. The cultures were washed with PBS to remove nonadherent cells. The resulting adherent cells were subjected to MTT assay. The enzyme activity obtained after culturing bone marrow cells in the presence of M-CSF was regarded as 100%. Samples were measured in three replicates and each experiment was repeated at least twice. Data are presented as a percentage of relative activity against the activity obtained from the culture with M-CSF alone and expressed as p Ͻ 0.01; related to the activity of the ,ءء ;p Ͻ 0.05 ,ء .mean Ϯ SEM culture in the presence of M-CSF alone. C, Number of TRAP-positive cells. Bone marrow cells were treated with M-CSF (lane1); M-CSF and ␣ ␣ ␣ TNF- (lane 2); M-CSF, TNF- , and IL-12 (lane 3); and M-CSF, TNF- , FIGURE 9. Effect of IL-12 on Fas and FasL expression in the adherent ␮ IL-12, and anti-FasL Ab (1 g/ml) (lane 4) for 4 days. Cells were washed cells and the nonadherent cells of bone marrow cells. Bone marrow cells to remove nonadherent cells and were then fixed and stained for TRAP. were treated with M-CSF (lane 1); M-CSF and TNF-␣ (lane 2); and Ͻ ء Ϯ Results were expressed as mean SEM of three cultures. , p 0.05; M-CSF and IL-12 (lane 3) for 3 days. A, RT-PCR. Cells were separated ␣ Ͻ ءء , p 0.01; related to the number of the culture with M-CSF and TNF- . into adherent cells and nonadherent cells. RNA was isolated from the ad- ␣ M-CSF, TNF- , and IL-12 were used at 50 ng/ml. herent cells and nonadherent cells and subjected to RT-PCR analysis. PCR amplification of the RNA samples without the reverse transcriptase treatment yielded no detectable fragments with either primer pair. B, Flow cytometry. Flow cytometry was performed using a FACScan flow cytom- was consistent with that in viability of the adherent cells, suggest- eter for detection of Fas and FasL on the cell surface. Mouse mAbs raised ing that anti-FasL Abs inhibited apoptosis of all the adherent cells, against Fas and FasL were incubated for 15 min with the adherent cells and including TRAP-positive cells in the culture. These results indi- nonadherent cells. After washing with PBS, cells were incubated with cated that apoptosis of the adherent cells might be mainly caused FITC-conjugated donkey anti-mouse Ab for 30 min, washed with PBS, by Fas/FasL interaction. diluted with 20 ml of PBS, and subjected to FACS analysis. The Journal of Immunology 4737 marrow macrophages without osteoblasts/stromal cells. They sug- lines of evidence for induction of FasL expression by IL-12 have gested in the study that even if a small number of stromal cells been provided. Yu et al. (46) showed that dendritic cell-derived were present in the preparation of bone marrow macrophages, they IL-12 is involved in up-regulation of FasL on NK cells leading to would not support osteoclast formation. In contrast, another group cell death. Leite-de-Moraes et al. (47) showed that IL-18 and IL-12 indicated that TNF-␣ alone, at any concentration, fails to induce up-regulate FasL expression in NKT cells. Activated NKT cells the differentiation of murine osteoclast precursors and that, rather, have the ability to kill their target cells by perforin- or FasL-de- TNF-␣ dramatically enhances in vitro osteoclastogenesis primed pendent mechanisms, resulting in prevention of metastasis (48– by a low dose of RANKL that is insufficient to induce osteoclast 50). Dao et al. (51) have reported that IL-12 enhances the FasL- formation (32). In this study, we also examined the effect of mediated death of Th1 cells. FasL, expressed on activated T cells, TNF-␣ on osteoclastogenesis. In cultures of bone marrow macro- plays a central role in regulating the immune response by inducing phages with TNF-␣ and M-CSF, TNF-␣ increased the number of apoptosis in activated lymphocytes through binding of FasL to its TRAP-positive cells in a dose-dependent manner, confirming that receptor, Fas. In this study, IL-12 up-regulated FasL transcription TNF-␣ was able to induce differentiation into osteoclasts under and induced FasL expression on the surfaces of the nonadherent these conditions. However, we cannot exclude the possibility of cells in bone marrow cell cultures in the presence of M-CSF. How- contamination with very small amounts of RANKL that would be ever, TNF-␣ failed to induce FasL in the cells. Therefore, both produced from trace remaining osteoblasts/stromal cells in this IL-12 and TNF-␣ were necessary to induce apoptosis in this cul- culture. Additional experiments are necessary to clarify this aspect. ture system. The apoptosis seems to be induced by Fas/FasL in- IL-12 has been reported to have positive and negative effects on teraction; however, IL-12 may also trigger some other apoptotic apoptosis of cells. IL-12 induces apoptosis of human osteosarcoma mechanisms because the inhibitory effect of anti-FasL Ab on the and breast cancer cells (33) and suppresses tumor metastases in apoptosis was incomplete. Downloaded from both liver and lung by induction of a TNF-related apoptosis-in- Target cells for IL-12 have been reported to include T cells ducing ligand-dependent apoptosis (34). In contrast, IL-12 inhibits (21–25), NK cells (21, 22, 46), NKT cells (47), B cells (52), den- UV-induced apoptosis of keratinocytes (35) and apoptosis of naive dritic cells (53), and macrophages (54). In this study, we found that allogeneic T cells caused by liver nonparenchymal cells in vitro IL-12 influenced nonadherent cells in bone marrow cell cultures (36). These bilateral effects indicated that IL-12 may affect several and elicited FasL from the cells, suggesting that dendritic cells and aspects of cell biology. In this study, we found that IL-12 could macrophages are excluded from the target cells. Apoptotic alter- http://www.jimmunol.org/ induce apoptosis of bone marrow cells in combination with ations were not observed in bone marrow macrophages that were TNF-␣. The induction of apoptosis of bone marrow cells by IL-12 cocultured with T cells isolated from spleen cells in the presence seems to be novel because it requires both IL-12 and TNF-␣. of M-CSF, TNF-␣, and IL-12 (H. Kitaura and K. Nakayama, un- When bone marrow cells were cultured in the presence of published observations). In addition, when whole bone marrow M-CSF, TNF-␣, and IL-12 for 24 h, viability of bone marrow cells cells from nude mice that have very few mature T cells because of was similar to that in the cultures with M-CSF alone, M-CSF and a genetic thymus defect were cultured in the presence of M-CSF, TNF-␣, and M-CSF and IL-12. However, when bone marrow cells TNF-␣, and IL-12, the cells lapsed into apoptosis as did those from were cultured under these conditions for 48, 72, and 96 h, viability normal mice (H. Kitaura and K. Nakayama, unpublished observa- by guest on October 6, 2021 of the cells was gradually decreased, whereas we found no signif- tions). These unpublished observations indicate that T cells may icant change of viability of bone marrow cells in the culture with not be involved in the target cells for IL-12 in the bone marrow cell M-CSF alone, M-CSF and TNF-␣, or M-CSF and IL-12. It is culture. Additional experiments are necessary to find out the target plausible that the decrease of the number of the adherent cells in cells for IL-12. Experiments concerning NK cells and B cells are the presence of M-CSF, TNF-␣, and IL-12 may result from apo- now in progress. ptosis of the cells rather than loss of cell adherence because via- IL-12 can inhibit osteoclast formation in mouse spleen cells in bility of whole bone marrow cells was decreased under the same vitro (27). We have recently found that IL-12 can also inhibit conditions. osteoclast formation in mouse bone marrow cells treated with Many cytokines such as IL-3, IL-6, and GM-CSF have been M-CSF and RANKL (N. Nagata, H. Kitaura, and K. Nakayama, shown not only to stimulate growth and differentiation of hema- unpublished observations). In this study, IL-12 was found to in- topoietic progenitor cells, but also to have specific viability-pro- duce apoptosis of the adherent cells in TNF-␣-mediated osteoclast moting effects by which they can inhibit apoptosis (37). Other formation of mouse bone marrow cells, suggesting that IL-12 can cytokines including TGF-␤, TNF-␣, and IFN-␥ mediate predom- inhibit osteoclast formation that is related to both physiological inantly growth-suppressing effects and are able to promote apo- bone resorption induced by RANKL and pathological bone resorp- ptosis of hematopoietic progenitor cells (37, 38). Fas and FasL are tion induced by TNF-␣. critically involved in regulation of the immune system (31). Fas is a transmembrane protein of the TNF death receptor family ex- Acknowledgments pressed by a variety of tissues and several mature hematopoietic We thank Dr. Akira Yamaguchi for his valuable advice, discussions, and lineages such as T and B lymphocytes (39, 40), monocytes, and critical reading of the manuscript. granulocytes at different stages of maturation (41–43). 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