Inflammation ( # 2013) DOI: 10.1007/s10753-013-9782-0
Effect of IL-15 and Natural Killer Cells on Osteoclasts and Osteoblasts in a Mouse Coculture Hiroaki Takeda,1 Takeshi Kikuchi,1,2 Kyoko Soboku,1 Iichiro Okabe,1 Hiroki Mizutani,1 Akio Mitani,1 Yuichi Ishihara,1 and Toshihide Noguchi1
Abstract—This study analyzes the effect of interleukin-15 (IL-15) on osteoclast formation using a coculture of mouse osteoblasts and bone marrow cells (BMCs) stimulated with prostaglandin E2 (PGE2), which both have important role in rheumatoid arthritis (RA) and periodontal disease (PD). BMCs isolate lacking T (BMT−) or NK (BMNK−) cells, BMCs with no cells removed (BMT+NK+), purified NK cells, and purified T cells were each cocultured with osteoblasts in the presence or absence of PGE2 and/or IL15. The number of both osteoclasts and osteoblasts was decreased by IL-15 in a dose-dependent manner in BMT+NK+, BMT−. However, the reductions were improved in BMNK−. The expression of caspase3 in osteoblasts cocultured with NK cells was increased in a dose-dependent manner by IL-15. IL-15 stimulates apoptosis of osteoblasts via activation of NK cells. Since osteoblasts have an important role in bone formation, IL-15 may be an inflammatory bone destructive factor in RA and PD. KEY WORDS: inflammation; bone metabolism; osteoblast; osteoclast; apoptosis.
INTRODUCTION Prevalent chronic inflammatory disorders that affect bone structures include rheumatoid arthritis (RA) and periodontal disease (PD). Chronic inflammation is the key mediator of local and systemic bone loss in RA and PD. In RA and PD patients, cytokines are rapidly synthesized, as evidenced by increased levels of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 at sites of inflammation [1–3]. It is well documented that these proinflammatory cytokines promote bone resorption, acting on osteoblasts and stimulating osteoclastogenesis through upregulation of the receptor activator of NFκB ligand (RANKL) and downregulation of osteoprotegerin in osteoblasts [4]. Recently, IL-15, a proinflammatory cytokine, was implicated as having an important role in RA and PD disease progression [5–7].
1
Department of Periodontology, School of Dentistry, Aichi Gakuin University, 2-11 Suemoridori, Chikusa-ku, Nagoya, Aichi 464-8651, Japan 2 To whom correspondence should be addressed at Department of Periodontology, School of Dentistry, Aichi Gakuin University, 2-11 Suemoridori, Chikusa-ku, Nagoya, Aichi 464-8651, Japan. E-mail: [email protected]
IL-15 was simultaneously discovered by two different laboratories in 1994 and characterized as a T cell growth factor [8, 9]. The heterotrimeric receptor of IL-15 has homologous to the IL-2 receptor: both possess the IL-2R/ IL-15Rβ (CD122) and common gamma (γc) chain (CD132) [10]. IL-15 shares most of the biological activities of IL-2 on several types of lymphocytes, including regulation of the proliferation and activation of T cells, natural killer (NK) cells, and B cells [8–12]. There are reports that the expression of IL-15 in RA and PD patients is higher than in healthy individuals [5–7]. IL-15 was detected at concentrations up to 1,200 ng/mL in synovial fluid of RA patients [5], and its average concentration was 21.650 pg/ uL in gingival crevicular fluid from gingivitis patients [13]. Meanwhile, neutralization of IL-15 inhibits inflammation in RA patients [14]. IL-15 is a pleiotropic proinflammatory cytokine that appears to have a role in the pathological bone loss that occurs in diseases such as RA and PD. IL-15 may be a putative modulator of osteoclast differentiation, a hypothesis supported by the observations that IL-15 stimulates the formation of multinucleated osteoclast-like cells in rat bone marrow in vitro [15] and that neutralization of IL-15 prevents bone destruction in collagen-induced arthritis [16]. IL-15 produced by RA T cells can also induce osteoclastogenesis in cocultured autologous monocytes
0360-3997/13/0000-0001/0 # 2013 Springer Science+Business Media New York
Takeda, Kikuchi, Soboku, Okabe, Mizutani, Mitani, Ishihara, and Noguchi [17]. Meanwhile, compared with wild type, osteoclastogenesis and osteoclast function are reduced in IL-15Rdeficient spleen and bone marrow cells [18]. Bone mineral density is increased in IL-15R-deficient mice, a phenomenon not reversed by ovariectomy [18]. Serum levels of tartrate-resistant acid phosphatase (TRAP) and osteocalcin were lower in IL-15R-deficient mice, consistent with a low bone turnover in the absence of IL-15 signaling [18]. These observations suggest that IL-15 is closely associated with inflammatory bone destruction and stimulates osteoclast differentiation. Thus, it was hypothesized that IL-15 is involved in osteoclastogenesis through its effect(s) on osteoblastic cells. We investigated the effect of IL-15 on osteoclast formation using a coculture of mouse osteoblasts and bone marrow cells (BMCs) stimulated with PGE2, which has an important role in inflammatory bone diseases such as RA and PD.
MATERIALS AND METHODS Isolation of Osteoblasts and BMCs Osteoblasts were isolated from the calvariae of 1–3day-old ddY mice (CKS Inc., Aichi, Japan) [19]. The calvariae were digested in 10 mL of α-minimum essential medium (α-MEM) (Gibco-BRL, Grand Island, NY, USA) containing 0.1 % collagenase (Wako Pure Chemicals, Osaka, Japan) and 0.2 % dispase (Godo Shusei Inc., Tokyo, Japan) for 10 min at 37 °C with vigorous shaking and then centrifuged at 300×g for 5 min. The first supernatant was discarded, after which 10 mL of fresh collagenase/dispase enzyme solution was added and the calvariae were incubated for a further 10 min. This digestion procedure was repeated four times, with the supernatants from the final three cycles collected. The cells isolated in this combined supernatant were designated as osteoblasts and were cultured for 3–4 days in α-MEM containing 10 % fetal bovine serum (FBS) (MP Biomedicals, Solon, OH, USA) and antibiotic solution (100 U/mL penicillin, 100 μg/mL streptomycin; Gibco-BRL, Grand Island, NY, USA) in a humidified atmosphere of 5 % CO2 at 37 °C, with the medium changed after 3 days. The BMCs were collected from 4–6-week-old ddY mice (CKS Inc., Aichi, Japan) [19]. The ends of the tibiae were removed and the bone marrow cavity was flushed by slowly injecting α-MEM into one end using a 25-gauge needle. The BMCs were washed by centrifugation and
Fig. 1. Effects of IL-15 on PGE2-induced osteoclast formation. Osteoblasts from mouse calvaria were cocultured with BMT+NK+, BMT−, or BMNK− cells and then treated with PGE2 (1×10−6 M) in the absence or presence of the indicated concentrations of IL-15 for 6 days. Cells were then stained for TRAP and observed at × 100 magnification under a light microscope (a–c). Osteoclasts (designated as TRAP-positive multinucleated cells containing more than three nuclei) were counted in each culture, with mean values (±standard deviation) shown in d–f. Statistical analysis was by one-way ANOVA followed by Tukey’s post hoc multiple comparisons test. Single asterisk and double asterisks denote a statistically significant difference (p
Effect of IL-15 and Natural Killer Cells on Osteoclasts and Osteoblasts in a Mouse Coculture Hiroaki Takeda,1 Takeshi Kikuchi,1,2 Kyoko Soboku,1 Iichiro Okabe,1 Hiroki Mizutani,1 Akio Mitani,1 Yuichi Ishihara,1 and Toshihide Noguchi1
Abstract—This study analyzes the effect of interleukin-15 (IL-15) on osteoclast formation using a coculture of mouse osteoblasts and bone marrow cells (BMCs) stimulated with prostaglandin E2 (PGE2), which both have important role in rheumatoid arthritis (RA) and periodontal disease (PD). BMCs isolate lacking T (BMT−) or NK (BMNK−) cells, BMCs with no cells removed (BMT+NK+), purified NK cells, and purified T cells were each cocultured with osteoblasts in the presence or absence of PGE2 and/or IL15. The number of both osteoclasts and osteoblasts was decreased by IL-15 in a dose-dependent manner in BMT+NK+, BMT−. However, the reductions were improved in BMNK−. The expression of caspase3 in osteoblasts cocultured with NK cells was increased in a dose-dependent manner by IL-15. IL-15 stimulates apoptosis of osteoblasts via activation of NK cells. Since osteoblasts have an important role in bone formation, IL-15 may be an inflammatory bone destructive factor in RA and PD. KEY WORDS: inflammation; bone metabolism; osteoblast; osteoclast; apoptosis.
INTRODUCTION Prevalent chronic inflammatory disorders that affect bone structures include rheumatoid arthritis (RA) and periodontal disease (PD). Chronic inflammation is the key mediator of local and systemic bone loss in RA and PD. In RA and PD patients, cytokines are rapidly synthesized, as evidenced by increased levels of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 at sites of inflammation [1–3]. It is well documented that these proinflammatory cytokines promote bone resorption, acting on osteoblasts and stimulating osteoclastogenesis through upregulation of the receptor activator of NFκB ligand (RANKL) and downregulation of osteoprotegerin in osteoblasts [4]. Recently, IL-15, a proinflammatory cytokine, was implicated as having an important role in RA and PD disease progression [5–7].
1
Department of Periodontology, School of Dentistry, Aichi Gakuin University, 2-11 Suemoridori, Chikusa-ku, Nagoya, Aichi 464-8651, Japan 2 To whom correspondence should be addressed at Department of Periodontology, School of Dentistry, Aichi Gakuin University, 2-11 Suemoridori, Chikusa-ku, Nagoya, Aichi 464-8651, Japan. E-mail: [email protected]
IL-15 was simultaneously discovered by two different laboratories in 1994 and characterized as a T cell growth factor [8, 9]. The heterotrimeric receptor of IL-15 has homologous to the IL-2 receptor: both possess the IL-2R/ IL-15Rβ (CD122) and common gamma (γc) chain (CD132) [10]. IL-15 shares most of the biological activities of IL-2 on several types of lymphocytes, including regulation of the proliferation and activation of T cells, natural killer (NK) cells, and B cells [8–12]. There are reports that the expression of IL-15 in RA and PD patients is higher than in healthy individuals [5–7]. IL-15 was detected at concentrations up to 1,200 ng/mL in synovial fluid of RA patients [5], and its average concentration was 21.650 pg/ uL in gingival crevicular fluid from gingivitis patients [13]. Meanwhile, neutralization of IL-15 inhibits inflammation in RA patients [14]. IL-15 is a pleiotropic proinflammatory cytokine that appears to have a role in the pathological bone loss that occurs in diseases such as RA and PD. IL-15 may be a putative modulator of osteoclast differentiation, a hypothesis supported by the observations that IL-15 stimulates the formation of multinucleated osteoclast-like cells in rat bone marrow in vitro [15] and that neutralization of IL-15 prevents bone destruction in collagen-induced arthritis [16]. IL-15 produced by RA T cells can also induce osteoclastogenesis in cocultured autologous monocytes
0360-3997/13/0000-0001/0 # 2013 Springer Science+Business Media New York
Takeda, Kikuchi, Soboku, Okabe, Mizutani, Mitani, Ishihara, and Noguchi [17]. Meanwhile, compared with wild type, osteoclastogenesis and osteoclast function are reduced in IL-15Rdeficient spleen and bone marrow cells [18]. Bone mineral density is increased in IL-15R-deficient mice, a phenomenon not reversed by ovariectomy [18]. Serum levels of tartrate-resistant acid phosphatase (TRAP) and osteocalcin were lower in IL-15R-deficient mice, consistent with a low bone turnover in the absence of IL-15 signaling [18]. These observations suggest that IL-15 is closely associated with inflammatory bone destruction and stimulates osteoclast differentiation. Thus, it was hypothesized that IL-15 is involved in osteoclastogenesis through its effect(s) on osteoblastic cells. We investigated the effect of IL-15 on osteoclast formation using a coculture of mouse osteoblasts and bone marrow cells (BMCs) stimulated with PGE2, which has an important role in inflammatory bone diseases such as RA and PD.
MATERIALS AND METHODS Isolation of Osteoblasts and BMCs Osteoblasts were isolated from the calvariae of 1–3day-old ddY mice (CKS Inc., Aichi, Japan) [19]. The calvariae were digested in 10 mL of α-minimum essential medium (α-MEM) (Gibco-BRL, Grand Island, NY, USA) containing 0.1 % collagenase (Wako Pure Chemicals, Osaka, Japan) and 0.2 % dispase (Godo Shusei Inc., Tokyo, Japan) for 10 min at 37 °C with vigorous shaking and then centrifuged at 300×g for 5 min. The first supernatant was discarded, after which 10 mL of fresh collagenase/dispase enzyme solution was added and the calvariae were incubated for a further 10 min. This digestion procedure was repeated four times, with the supernatants from the final three cycles collected. The cells isolated in this combined supernatant were designated as osteoblasts and were cultured for 3–4 days in α-MEM containing 10 % fetal bovine serum (FBS) (MP Biomedicals, Solon, OH, USA) and antibiotic solution (100 U/mL penicillin, 100 μg/mL streptomycin; Gibco-BRL, Grand Island, NY, USA) in a humidified atmosphere of 5 % CO2 at 37 °C, with the medium changed after 3 days. The BMCs were collected from 4–6-week-old ddY mice (CKS Inc., Aichi, Japan) [19]. The ends of the tibiae were removed and the bone marrow cavity was flushed by slowly injecting α-MEM into one end using a 25-gauge needle. The BMCs were washed by centrifugation and
Fig. 1. Effects of IL-15 on PGE2-induced osteoclast formation. Osteoblasts from mouse calvaria were cocultured with BMT+NK+, BMT−, or BMNK− cells and then treated with PGE2 (1×10−6 M) in the absence or presence of the indicated concentrations of IL-15 for 6 days. Cells were then stained for TRAP and observed at × 100 magnification under a light microscope (a–c). Osteoclasts (designated as TRAP-positive multinucleated cells containing more than three nuclei) were counted in each culture, with mean values (±standard deviation) shown in d–f. Statistical analysis was by one-way ANOVA followed by Tukey’s post hoc multiple comparisons test. Single asterisk and double asterisks denote a statistically significant difference (p
