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The Journal of Immunology, 2002, 169: 4732-4738.
Copyright © 2002 by The American Association of Immunologists

Effect of IL-12 on TNF-{alpha}-Mediated Osteoclast Formation in Bone Marrow Cells: Apoptosis Mediated by Fas/Fas Ligand Interaction1

Hideki Kitaura2,*, Noriko Nagata*,{dagger}, Yuji Fujimura*,{dagger}, Hitoshi Hotokezaka*, Noriaki Yoshida* and Koji Nakayama{dagger}

* Divisions of Orthodontic and Biomedical Engineering and {dagger} Microbiology and Oral Infection, Department of Developmental and Reconstructive Medicine, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, it has been found that differentiation into osteoclasts is induced by TNF-{alpha}. In this study, we investigated the effect of IL-12 on TNF-{alpha}-mediated osteoclastogenesis. When mouse bone marrow cells were cultured with TNF-{alpha}, osteoclast-like cells were formed. When they were cultured with both TNF-{alpha} 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-{alpha} 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 the adherent cells was markedly inhibited by anti-Fas ligand (FasL) Ab. RT-PCR and FACS analyses revealed that TNF-{alpha} 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-{alpha} 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-{alpha}-induced Fas on the adherent cells and IL-12-induced FasL on the nonadherent cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bone resorption is controlled by osteoclasts that are differentiated from hematopoietic stem cells (1, 2, 3). Bone marrow contains only a small number of mature osteoclasts in a nonproliferative state because mature osteoclasts are multinuclear giant cells formed by fusion of mononuclear cells. Formation of mature osteoclasts requires two cellular factors: M-CSF and the receptor activator of NF-{kappa}B ligand (RANKL).3 M-CSF is indispensable for proliferation and/or differentiation of osteoclast precursors (4, 5). The osteopetrotic op/op mice are extremely deficient in osteoclasts and macrophages. This deficiency is caused by the absence of functional M-CSF and can be cured by injections of M-CSF (6). In contrast, RANKL has been identified as a ligand for RANK, an immunoresponsive receptor on dendritic cells (7). RANKL, which is also called TNF-related activation-induced cytokine (8), osteoclast differentiation factor (9), and osteoprotegerin ligand (10), can cause differentiation from osteoclast precursors into mature osteoclasts in the presence of M-CSF in in vitro culture systems. RANKL-deficient mice show severe osteopetrosis and lack osteoclasts completely (11).

Kobayashi et al. (12) have reported that TNF-{alpha} induces osteoclast-like cells from M-CSF-dependent bone marrow-derived macrophages in vitro. TNF-{alpha} is known to play a major role in host defense, and it exerts proinflammatory activities through various cells including mononuclear phagocytes, in which it is responsible for the activation of bactericidal/cytocidal systems (13, 14). TNF-{alpha} is involved in differentiation into both osteoclasts and macrophages, although their biological roles seem quite different.

TNF-{alpha} is pleiotropic, which has a variety of often opposing biological effects in a cell-specific manner. As one of its most perplexing properties, TNF-{alpha} promotes cell survival in certain conditions and cell death in others (15). The findings that TNF-{alpha} recognizes two receptors on cell surfaces (type 1 or p55 and type 2 or p75 receptors) and that each receptor is capable of distinct intracellular signaling (15) have substantially deepened understanding of the complex activities of the cytokine. TNF-{alpha}-induced osteoclast recruitment is probably central to the pathogenesis of disorders with inflammatory osteolysis such as periodontal disease (16) and periprosthetic bone loss (17). In fact, TNF-{alpha} is shown to be involved in the causes of postmenopausal osteoporosis (18, 19).

IL-12 is a heterodimeric 70-kDa protein that is composed of two subunits (p35 and p40) linked by a disulfide bond. IL-12 has been found to have the ability to induce maturation of cytotoxic T lymphocytes and to enhance production of IFN-{gamma} in NK cells (20, 21, 22). IL-12 has also been shown to have a pivotal role in Th1-dominant immune responses such as host defense responses against intracellular pathogens (23, 24, 25).

In a recent study, mouse and human osteoblasts in vitro infected with Staphylococcus aureus have been found to express high levels of IL-12 (26). More recently, Horwood and colleagues (27, 28) have found that IL-12 can inhibit osteoclast formation in spleen cell cultures in vitro and that the IL-12-mediated inhibition of osteoclast formation is T cell dependent, like the inhibitory action of IL-18.

In this study, we investigated the effect of IL-12 on TNF-{alpha}-induced osteoclastogenesis in bone marrow cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and reagents

Five-week-old male ddY mice were purchased from Seac Yoshitomi (Fukuoka, Japan). Recombinant human M-CSF was purchased from Yoshitomi Pharmaceutical (Tokyo, Japan) and recombinant mouse TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN). Recombinant mouse IL-12 was obtained from Wako Pure Chemical (Osaka, Japan), and anti-Fas and anti-Fas ligand (FasL) Abs were from BD PharMingen (San Diego, CA).

TNF-{alpha}-induced osteoclast formation in bone marrow cells

The femora and tibiae of mice were aseptically removed and dissected free of adhering tissues. The bone ends were cut off by scissors and the marrow cavity was flushed out by slow injection of {alpha}-MEM (Sigma-Aldrich, Tokyo, Japan) at one end of the bone using a sterile needle to collect bone marrow cells. After washing with {alpha}-MEM, cells were incubated in culture medium ({alpha}-MEM containing 10% FBS, 100 IU/ml penicillin G (Meiji Seika, Tokyo, Japan), and 100 µg/ml streptomycin (Meiji Seika)). Whole bone marrow cells were cultured at 2 x 106 cells/ml in a 48-well plate in the presence of M-CSF (50 ng/ml) and TNF-{alpha} (50 ng/ml) for 1–5 days without medium change in the cultures. M-CSF, TNF-{alpha}, and IL-12 were used at a final concentration of 50 ng/ml in this study, except in the experiments with indicated concentrations.

Tartrate-resistant acid phosphatase (TRAP) staining

Cultured cells were fixed with 4% paraformaldehyde for 30 min and then 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 tartrate.

Bone marrow-derived macrophages

Bone marrow cells were incubated in culture medium supplemented with M-CSF (100 ng/ml) at 1 x 107 cells per 10 ml in a 10-cm culture dish. After 3-day culture, cells were washed vigorously with PBS twice to remove nonadherent cells, harvested by pipetting with 0.02% EDTA in PBS, and seeded at 1 x 106 cells per 10 ml in a 10-cm dish. After an additional 3-day culture, cells were harvested. We used these cells as bone marrow macrophages in this study (29).

Cell viability assay

Cell viability was determined by the MTT assay. Cultures were washed with PBS twice to remove nonadherent cells. The adherent cells were cultured in 1 ml of culture medium in a well. Ten microliters of MTT (10 mg/ml) were added to each well, and the mixture was incubated for 4 h at 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 crystals. 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.

RNA preparation and analysis

Total RNA was isolated from adherent cells and nonadherent cells that had been cultured for 72 h using TRIzol reagent (Life Technologies, Grand Island, NY). For RT-PCR analysis, 2 µg of RNA samples were dissolved in 20 µl of the reaction mixture (50 mM Tris · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.1% Triton X-100, 10 mM DTT, 0.5 mM deoxynucleotide triphosphates, 5 µM random hexamer primers (Promega, Madison, WI), and 40 U/µl Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA)) and were incubated at 42°C for 50 min followed by incubation at 70°C for 15 min. The resulting cDNA was then diluted to 100 µl with distilled water. PCR amplification was performed in a reaction mixture (50 µl) containing the cDNA solution (5 µl), 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.1% Triton X-100, 0.2 mM deoxynucleotide triphosphates, 1 U of Taq polymerase (Wako Nippon Gene, Tokyo, Japan), and 1 mM appropriate primers. The primers used were as follows: sense, 5'-TCCTGTGGCATCCATGAAACT-3', and antisense, 5'-CTTCGTGAACGCCACGTGCTA-3', for {beta}-actin; sense, 5'-TTGCTGTCAACCATGCCAAC-3', and antisense, 5'-ACGTGAACCATAAGACCCAG-3', for Fas; sense, 5'-ATCCCTCTGGAATGGGAAGA-3', and antisense, 5'-CCATATCTGTCCAGTAGTGC-3', for FasL. The conditions for amplification were as follows: one cycle (93°C, 3 min), 30 cycles (93°C, 1 min; 55°C, 1 min; 72°C, 2 min), and one cycle (72°C, 7 min) for {beta}-actin and FasL; and one cycle (93°C, 3 min), 30 cycles (93°C, 1 min; 63°C, 1 min; 72°C, 2 min), and one cycle (72°C, 7 min) for Fas.

FACS and flow cytometry

The adherent and nonadherent cells of bone marrow cells were incubated for 15 min with the monoclonal mouse Abs raised against Fas and FasL. After being washed with PBS, cells were incubated with FITC-conjugated donkey anti-mouse Ab (Sigma-Aldrich) for 30 min, washed, diluted with 20 ml of PBS, and subjected to FACS analysis. Samples were analyzed using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ) for detection of Fas and FasL.

Statistical analysis

Differences between data were analyzed with the Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro osteoclast differentiation by TNF-{alpha}

Initially, we examined whether TNF-{alpha} could induce osteoclast differentiation in an in vitro culture system of bone marrow cells. TRAP-positive cells were found after culturing bone marrow cells in the presence of 100 ng/ml TNF-{alpha} for 3 days, although no TRAP-positive cells were detected in the cultures with TNF-{alpha} of concentrations lower than 1 ng/ml or in the culture in the presence of M-CSF alone (Figs. 1Go and 2Go). The number of TRAP-positive cells increased along with increase of TNF-{alpha} (10–100 ng/ml) in 4- and 5-day cultures (Fig. 1Go). Most of the TRAP-positive cells were mononucleated until day 5. When M-CSF and TNF-{alpha} were added again to the cultures at day 5, multinucleated cells appeared in 7-day culture (data not shown).



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  		FIGURE 1. Effect of TNF-{alpha} on formation of TRAP-positive cells in bone marrow cells. Bone marrow cells of ddY mice were cultured with 50 ng/ml M-CSF and various concentrations of TNF-{alpha} for 1–5 days. Cells were washed to remove nonadherent cells and were then fixed and stained for TRAP. The number of TRAP-positive cells was scored. Results were expressed as mean ± SEM of three cultures.

 


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  		FIGURE 2. Effect of IL-12 on TNF-{alpha}-induced formation of TRAP-positive cells in bone marrow cells. Bone marrow cells were treated with M-CSF (A); M-CSF and TNF-{alpha} (B); M-CSF, TNF-{alpha}, 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-{alpha}-induced osteoclast formation from bone marrow cells

To examine the effect of IL-12 on TNF-{alpha}-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-{alpha}. When IL-12 was added to the culture with M-CSF and TNF-{alpha}, the number of TRAP-positive cells was markedly decreased in a dose-dependent fashion (Figs. 2Go and 3Go). The number of adherent cells was also decreased and the remaining adherent cells showed cytopathic changes (Fig. 2GoC). 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-{alpha} (Fig. 2GoD). We examined these cells for viability using MTT assay. When bone marrow cells were cultured in the presence of M-CSF and TNF-{alpha}, 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-{alpha}, and IL-12, viability of the cells was decreased to <10\% of that with M-CSF alone after culturing for 3 days (Fig. 4>A). Decrease of viability was dependent on the concentration of IL-12 (Fig. 4GoB). 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. 4GoA). These results indicated that death of bone marrow cells was induced by culturing with a combination of M-CSF, TNF-{alpha}, and IL-12.



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  		FIGURE 3. Effect of IL-12 on the number of TRAP-positive cells in bone marrow cells in the presence of M-CSF and TNF-{alpha}. Bone marrow cells of ddY mice were cultured with M-CSF (50 ng/ml), TNF-{alpha} (50 ng/ml), and an indicated concentration of IL-12 for 4 days. After washing with PBS to remove nonadherent cells, cells were fixed and the number of TRAP-positive cells was scored. Results were expressed as mean ± SEM of three cultures. *, p < 0.05; **, p < 0.01; related to the activity of the culture without IL-12.

 


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  		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-{alpha} (lane 2); M-CSF, TNF-{alpha}, 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 with M-CSF alone and expressed as mean ± SEM. *, p < 0.05; **, p < 0.01; related to the activity of the culture in the presence of M-CSF. B, IL-12 concentration. Bone marrow cells were treated with M-CSF (50 ng/ml), TNF-{alpha} (50 ng/ml), and an indicated concentration of IL-12 for 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 without IL-12 and expressed as mean ± SEM. *, p < 0.05; **, p < 0.01; related to the activity of the culture without IL-12.

 
Nuclear fragmentation

The morphology of nuclei of the adherent cells was investigated using the nuclear stain Hoechst 33342. The adherent cells in the culture with M-CSF alone, M-CSF and TNF-{alpha}, 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-{alpha}, and IL-12 (Fig. 5Go).



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  		FIGURE 5. Nuclear morphology of the adherent cells in bone marrow cells. Bone marrow cells were treated with M-CSF (A); M-CSF and TNF-{alpha} (B); M-CSF, TNF-{alpha}, and IL-12 (C); and M-CSF and IL-12 (D) for 4 days. M-CSF, TNF-{alpha}, and IL-12 were used at 50 ng/ml. After being washed with PBS to remove nonadherent cells, the resulting adherent cells were fixed and stained with Hoechst 33342.

 
DNA fragmentation

Fig. 6Go shows that DNA fragmentation resulting in DNA ladders was observed in the adherent cells in the culture with a combination of M-CSF, TNF-{alpha}, 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-{alpha}, or M-CSF and IL-12.



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  		FIGURE 6. Fragmentation of the chromosomal DNA of the adherent cells treated with M-CSF, TNF-{alpha}, and IL-12. Bone marrow cells were treated with M-CSF (lane 1); M-CSF and TNF-{alpha} (lane 2); M-CSF, TNF-{alpha}, and IL-12 (lane 3); and M-CSF and IL-12 (lane 4) for 4 days. M-CSF, TNF-{alpha}, and IL-12 were used at 50 ng/ml. Cells were washed with PBS to remove nonadherent cells. Chromosomal DNA was isolated from the adherent cells and subjected to electrophoresis on a 1.5% agarose gel containing ethidium bromide. DNA was visualized under UV light. MW, m.w. standard, {lambda}DNA-HindIII digest.

 
No effect of IL-12 on TNF-{alpha}-induced osteoclast formation from bone marrow macrophages

Because whole bone marrow cells consisted of various types of cells, it remained unclear whether IL-12 directly acted on precursors of osteoclasts. Therefore, direct actions of IL-12 on bone marrow macrophages enriched by treatment with M-CSF as osteoclast precursor cells were determined in TNF-{alpha}-induced osteoclast formation. As shown in Fig. 7Go, when bone marrow macrophages were treated with M-CSF and TNF-{alpha}, a number of TRAP-positive cells were formed. In contrast to the results of whole bone marrow cells, TRAP-positive cells were equally formed in the cultures of bone marrow macrophages treated with M-CSF and TNF-{alpha} in the presence or absence of IL-12. Apoptotic alterations such as atrophy and cytoclasis were not observed in bone marrow macrophages. These results indicated that apoptosis might not be induced by a direct action of IL-12 on precursors of osteoclasts, but by some factors induced by an action of IL-12 on nonosteoclast precursor cells in bone marrow cells.



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  		FIGURE 7. Effect of IL-12 on TNF-{alpha}-induced osteoclast formation from bone marrow macrophages. Bone marrow macrophages were treated with M-CSF (A), M-CSF and TNF-{alpha} (B), M-CSF and IL-12 (D), and M-CSF, TNF-{alpha}, and IL-12 (E) for 3 days; and M-CSF and IL-12 (C) and M-CSF, TNF-{alpha}, and IL-12 (F) for 5 days. M-CSF, TNF-{alpha}, and IL-12 were used at 50 ng/ml. Cells were fixed and stained for TRAP.

 
Induction of apoptosis of bone marrow cells in the presence of M-CSF, TNF-{alpha}, and IL-12 by Fas/FasL interaction

Fas/FasL interaction has been reported to induce apoptosis (31). Therefore, we investigated whether the induction of apoptosis of the adherent cells in bone marrow cells could be caused by Fas/FasL interaction. Apoptotic changes of bone marrow cells treated with a combination of M-CSF, TNF-{alpha}, and IL-12 were markedly inhibited by addition of anti-FasL to the culture (Fig. 8GoA). The decrease of viability of the adherent cells in bone marrow cells treated with a combination of M-CSF, TNF-{alpha}, 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-{alpha} or IL-12 (Fig. 8GoB). Also, the addition of anti-FasL Abs markedly inhibited the decrease of the number of TRAP-positive cells by treatment with M-CSF, TNF-{alpha}, and IL-12 (Fig. 8GoC). Although the number of TRAP-positive cells in the culture with M-CSF, TNF-{alpha}, IL-12, and anti-FasL did not reach the level of that in the culture with M-CSF and TNF-{alpha}, the rate of anti-FasL-mediated recovery in the number of TRAP-positive cells was consistent with that in viability of the adherent cells, suggesting that anti-FasL Abs inhibited apoptosis of all the adherent cells, including TRAP-positive cells in the culture. These results indicated that apoptosis of the adherent cells might be mainly caused by Fas/FasL interaction.



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  		FIGURE 8. Inhibitory effect of anti-FasL Ab on IL-12/TNF-{alpha}-induced apoptosis of bone marrow cells. A, Microscopic observation. Bone marrow cells were treated with M-CSF (a); M-CSF and TNF-{alpha} (b); M-CSF, TNF-{alpha}, and IL-12 (c); and M-CSF, TNF-{alpha}, IL-12, and anti-FasL Ab (1 µg/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-{alpha} (lane 2); M-CSF, TNF-{alpha}, and IL-12 (lane 3); and M-CSF and IL-12 (lane 4) with or without anti-FasL Ab (1 µg/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 mean ± SEM. *, p < 0.05; **, p < 0.01; related to the activity of the 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-{alpha} (lane 2); M-CSF, TNF-{alpha}, and IL-12 (lane 3); and M-CSF, TNF-{alpha}, IL-12, and anti-FasL Ab (1 µg/ml) (lane 4) for 4 days. Cells were washed to remove nonadherent cells and were then fixed and stained for TRAP. Results were expressed as mean ± SEM of three cultures. *, p < 0.05; **, p < 0.01; related to the number of the culture with M-CSF and TNF-{alpha}. M-CSF, TNF-{alpha}, and IL-12 were used at 50 ng/ml.

 
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-{alpha}. Nonadherent cells in the cultures with M-CSF alone and M-CSF and TNF-{alpha} showed no FasL mRNA, whereas FasL mRNA was clearly detected in the nonadherent cells in the culture with M-CSF and IL-12 (Fig. 9GoA).



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  		FIGURE 9. Effect of IL-12 on Fas and FasL expression in the adherent cells and the nonadherent cells of bone marrow cells. Bone marrow cells were treated with M-CSF (lane 1); M-CSF and TNF-{alpha} (lane 2); and M-CSF and IL-12 (lane 3) for 3 days. A, RT-PCR. Cells were separated into adherent cells and nonadherent cells. RNA was isolated from the adherent 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 cytometer for detection of Fas and FasL on the cell surface. Mouse mAbs raised against Fas and FasL were incubated for 15 min with the adherent cells and nonadherent cells. After washing with PBS, cells were incubated with FITC-conjugated donkey anti-mouse Ab for 30 min, washed with PBS, diluted with 20 ml of PBS, and subjected to FACS analysis.

 
FACS analysis revealed that TNF-{alpha} 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-{alpha} could not induce FasL on the cell surfaces (Fig. 9GoB). Therefore, both IL-12 and TNF-{alpha} 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-{alpha}-induced Fas on the adherent cells and IL-12-induced FasL on the nonadherent cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 finding that the targeted disruption of the gene encoding RANKL in mice develops severe osteopetrosis with the complete absence of TRAP-positive cells in bone tissues (11). However, Kobayashi et al. (12) have recently found that TNF-{alpha} together with M-CSF induces formation of TRAP-positive osteoclast-like cells in bone marrow macrophages without osteoblasts/stromal cells. They suggested in the study that even if a small number of stromal cells were present in the preparation of bone marrow macrophages, they would not support osteoclast formation. In contrast, another group indicated that TNF-{alpha} alone, at any concentration, fails to induce the differentiation of murine osteoclast precursors and that, rather, TNF-{alpha} dramatically enhances in vitro osteoclastogenesis primed by a low dose of RANKL that is insufficient to induce osteoclast formation (32). In this study, we also examined the effect of TNF-{alpha} on osteoclastogenesis. In cultures of bone marrow macrophages with TNF-{alpha} and M-CSF, TNF-{alpha} increased the number of TRAP-positive cells in a dose-dependent manner, confirming that TNF-{alpha} was able to induce differentiation into osteoclasts under these conditions. However, we cannot exclude the possibility of contamination with very small amounts of RANKL that would be produced from trace remaining osteoblasts/stromal cells in this culture. Additional experiments are necessary to clarify this aspect.

IL-12 has been reported to have positive and negative effects on apoptosis of cells. IL-12 induces apoptosis of human osteosarcoma and breast cancer cells (33) and suppresses tumor metastases in both liver and lung by induction of a TNF-related apoptosis-inducing ligand-dependent apoptosis (34). In contrast, IL-12 inhibits UV-induced apoptosis of keratinocytes (35) and apoptosis of naive allogeneic T cells caused by liver nonparenchymal cells in vitro (36). These bilateral effects indicated that IL-12 may affect several aspects of cell biology. In this study, we found that IL-12 could induce apoptosis of bone marrow cells in combination with TNF-{alpha}. The induction of apoptosis of bone marrow cells by IL-12 seems to be novel because it requires both IL-12 and TNF-{alpha}.

When bone marrow cells were cultured in the presence of M-CSF, TNF-{alpha}, and IL-12 for 24 h, viability of bone marrow cells was similar to that in the cultures with M-CSF alone, M-CSF and TNF-{alpha}, and M-CSF and IL-12. However, when bone marrow cells were cultured under these conditions for 48, 72, and 96 h, viability of the cells was gradually decreased, whereas we found no significant change of viability of bone marrow cells in the culture with M-CSF alone, M-CSF and TNF-{alpha}, or M-CSF and IL-12. It is plausible that the decrease of the number of the adherent cells in the presence of M-CSF, TNF-{alpha}, and IL-12 may result from apoptosis of the cells rather than loss of cell adherence because viability of whole bone marrow cells was decreased under the same conditions.

Many cytokines such as IL-3, IL-6, and GM-CSF have been shown not only to stimulate growth and differentiation of hematopoietic progenitor cells, but also to have specific viability-promoting effects by which they can inhibit apoptosis (37). Other cytokines including TGF-{beta}, TNF-{alpha}, and IFN-{gamma} mediate predominantly growth-suppressing effects and are able to promote apoptosis of hematopoietic progenitor cells (37, 38). Fas and FasL are critically involved in regulation of the immune system (31). Fas is a transmembrane protein of the TNF death receptor family expressed by a variety of tissues and several mature hematopoietic lineages such as T and B lymphocytes (39, 40), monocytes, and granulocytes at different stages of maturation (41, 42, 43). Recently, it has become evident that apoptosis through Fas/FasL interaction also occurs in early hematopoietic progenitor cells, in which it might have a role in maintaining homeostasis of blood cells. Recent studies have shown that the proinflammatory cytokines IFN-{gamma} and TNF-{alpha} can induce Fas expression on the cells, although human CD34+ bone marrow cells do not normally express Fas on their surfaces (44, 45). In this study, TNF-{alpha} up-regulated Fas transcription, leading to Fas expression on the surfaces of the adherent cells in bone marrow cell cultures in the presence of M-CSF. However, IL-12 could not induce Fas in the cells. In contrast, several lines of evidence for induction of FasL expression by IL-12 have been provided. Yu et al. (46) showed that dendritic cell-derived IL-12 is involved in up-regulation of FasL on NK cells leading to cell death. Leite-de-Moraes et al. (47) showed that IL-18 and IL-12 up-regulate FasL expression in NKT cells. Activated NKT cells have the ability to kill their target cells by perforin- or FasL-dependent mechanisms, resulting in prevention of metastasis (48, 49, 50). Dao et al. (51) have reported that IL-12 enhances the FasL-mediated death of Th1 cells. FasL, expressed on activated T cells, plays a central role in regulating the immune response by inducing apoptosis in activated lymphocytes through binding of FasL to its receptor, Fas. In this study, IL-12 up-regulated FasL transcription and induced FasL expression on the surfaces of the nonadherent cells in bone marrow cell cultures in the presence of M-CSF. However, TNF-{alpha} failed to induce FasL in the cells. Therefore, both IL-12 and TNF-{alpha} were necessary to induce apoptosis in this culture system. The apoptosis seems to be induced by Fas/FasL interaction; however, IL-12 may also trigger some other apoptotic mechanisms because the inhibitory effect of anti-FasL Ab on the apoptosis was incomplete.

Target cells for IL-12 have been reported to include T cells (21, 22, 23, 24, 25), NK cells (21, 22, 46), NKT cells (47), B cells (52), dendritic cells (53), and macrophages (54). In this study, we found that IL-12 influenced nonadherent cells in bone marrow cell cultures and elicited FasL from the cells, suggesting that dendritic cells and macrophages are excluded from the target cells. Apoptotic alterations were not observed in bone marrow macrophages that were cocultured with T cells isolated from spleen cells in the presence of M-CSF, TNF-{alpha}, and IL-12 (H. Kitaura and K. Nakayama, unpublished observations). In addition, when whole bone marrow cells from nude mice that have very few mature T cells because of a genetic thymus defect were cultured in the presence of M-CSF, TNF-{alpha}, and IL-12, the cells lapsed into apoptosis as did those from normal mice (H. Kitaura and K. Nakayama, unpublished observations). These unpublished observations indicate that T cells may not be involved in the target cells for IL-12 in the bone marrow cell culture. Additional experiments are necessary to find out the target cells for IL-12. Experiments concerning NK cells and B cells are now in progress.

IL-12 can inhibit osteoclast formation in mouse spleen cells in vitro (27). We have recently found that IL-12 can also inhibit osteoclast formation in mouse bone marrow cells treated with M-CSF and RANKL (N. Nagata, H. Kitaura, and K. Nakayama, unpublished observations). In this study, IL-12 was found to induce apoptosis of the adherent cells in TNF-{alpha}-mediated osteoclast formation of mouse bone marrow cells, suggesting that IL-12 can inhibit osteoclast formation that is related to both physiological bone resorption induced by RANKL and pathological bone resorption induced by TNF-{alpha}.


   Acknowledgments
 
We thank Dr. Akira Yamaguchi for his valuable advice, discussions, and critical reading of the manuscript.


   Footnotes
 
1 This work was supported in part by a Grant-in-Aid (13771268) for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan. Back

2 Address correspondence and reprint requests to Dr. Hideki Kitaura, Divisions of Orthodontic and Biomedical Engineering, Department of Developmental and Reconstructive Medicine, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan. E-mail address: hide{at}dh.nagasaki-u.ac.jp Back

3 Abbreviations used in this paper: RANKL, receptor activator of NF-{kappa}B ligand; FasL, Fas ligand; TRAP, tartrate-resistant acid phosphatase. Back

Received for publication June 20, 2002. Accepted for publication August 22, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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