Abstract

Adiponectin is an adipokine with potent anti-inflammatory properties. We previously reported that a globular adiponectin (gAd) suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced nuclear factor-κB activity, suggesting an anti-inflammatory effect of gAd. In this study, we investigated whether gAd is able to modulate the effect of A. actinomycetemcomitans lipopolysaccharide on cytokine induction in a murine macrophage cell line (RAW 264). The phosphorylation of p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and IκB kinase α/β and the degradation of IκB, which were induced by A. actinomycetemcomitans lipopolysaccharide intoxication, were clearly reduced in gAd-pretreated RAW 264 cells compared with the untreated cells. Expression levels of tumor necrosis factor (TNF)-α and interleukin-10 (IL-10) mRNA were assessed by real-time PCR. Cell-free supernatants were collected after 12 h of stimulation and analyzed by enzyme-linked immunosorbent assay for TNF-α and IL-10. Pretreatment with gAd significantly inhibited the A. actinomycetemcomitans lipopolysaccharide-induced TNF-α mRNA expression and protein secretion. In contrast, pretreatment with gAd significantly enhanced the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 mRNA expression and protein secretion. These data suggest a mechanism for the anti-inflammatory activity of gAd in local inflammatory lesions, such as periodontitis.

Introduction

Obesity, defined as an increased mass of adipose tissues, may be a possible risk factor for periodontal disease (Saito et al., 1998; Linden et al., 2007; Saito & Shimazaki, 2007). Adipose tissues secrete several bioactive molecules called adipokines, for example, adiponectin, leptin, and resistin. The plasma levels of adiponectin correlate negatively with body mass index (Arita et al., 1999), and accumulating evidence indicates that plasma adiponectin counteracts inflammatory responses in metabolic and cardiovascular complications (Ouchi & Walsh, 2008). The secretion of inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) by lipopolysaccharide-stimulated porcine macrophages is reduced by adiponectin pretreatment (Wulster-Radcliffe et al., 2004). Furthermore, the release of anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist is increased by adiponectin treatment (Wolf et al., 2004). Recently, we reported that adiponectin negatively regulates the responses to Toll-like receptor ligands (Yamaguchi et al., 2005) and inhibits osteoclast formation stimulated by lipopolysaccharide from Aggregatibacter actinomycetemcomitans in RAW 264 cells (Yamaguchi et al., 2007).

Adiponectin can exist as full-length adiponectin (fAd) or a truncated form, globular adiponectin (gAd) (Kadowaki & Yamauchi, 2005). The fAd form consists of an N-terminal collagen-like region and a C-terminal C1q-like globular domain (Tilg & Moschen, 2006). The gAd form is converted from fAd by proteolytic cleavage, such as leukocyte elastase secreted from activated monocytes and/or neutrophils (Waki et al., 2005).

Aggregatibacter actinomycetemcomitans, a gram-negative oral bacterium, plays a crucial role in the development of periodontal disease, especially in the destructive stage involving rapid gingival and alveolar bone destruction (Sosroseno & Herminajeng, 1995; Meyer & Fives-Taylor, 1997). The lipopolysaccharide of A. actinomycetemcomitans is a potent inducer of proinflammatory mediator secretion in a variety of cells, including epithelial cells, fibroblasts, and macrophages (Wilson et al., 1996; O'Brien-Simpson et al., 2004; Bodet et al., 2006).

In this study, we investigated the effects of gAd treatment on immunological reactions in A. actinomycetemcomitans lipopolysaccharide-stimulated RAW 264 cells.

Materials and methods

Cells and reagents

Murine macrophage-like cells from the RAW 264 cell line (RCB0535; RIKEN Cell Bank, Ibaraki, Japan) were maintained in RPMI-1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Thermo Trace Ltd, Melbourne, Australia) and 50 µg mL−1 gentamicin. The cultures were maintained at 37 °C under 5% CO2.

Aggregatibacter actinomycetemcomitans lipopolysaccharide was extracted from lyophilized cells of A. actinomycetemcomitans strain Y4 using a hot phenol/water procedure, treated with nuclease, and washed extensively with pyrogen-free water by ultracentrifugation. The lipopolysaccharide preparation was purified by chromatography on Sephadex G-200 (GE Healthcare, Piscataway, NJ) equilibrated with 10 mM Tris-HCl (pH 8.0) containing 0.2 M sodium chloride, 0.25% (w/v) deoxycholate, 1 mM EDTA, and 0.02% (w/v) sodium azide (Yamaguchi et al., 2007).

Purification of recombinant protein

The glutathione S-transferase fusion vector pGEX-6P-1 (GE Healthcare) containing the globular domain of mouse adiponectin (gAd) gene was provided by Dr I. Shimomura (Osaka University, Osaka, Japan). Recombinant gAd was prepared as described previously (Yamaguchi et al., 2005). The concentration of endotoxin in the obtained gAd was 7.05 pg µg−1 protein as determined with limulus amebocyte lysate assay (Wako, Tokyo, Japan).

Real-time quantitative PCR

Total RNA from RAW 264 cells was isolated using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). The RNA samples were reverse transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The primer and fluorescent probe sets for β-actin (Mm00607939_s1), TNF-α (Mm00443258_m1), and IL-1β (Mm01336189_m1) were purchased from Applied Biosystems. Amplification and detection of the cDNA was accomplished using a StepOne® Real-Time PCR System (Applied Biosystems) with TaqMan Fast Universal PCR Master Mix (Applied Biosystems) in a total volume of 20 µL. The following primers were designed using primer express version 1.0 (Applied Biosystems): IL-10, forward 5′-GCC AAG CCT TAT CGG AAA TG-3′ and reverse 5′-CAC CCA GGG AAT TCA AAT GC-3′ (102-bp product); β-actin, forward 5′-GGT CAG AAG GAC TCC TAT GTG G-3′ and reverse 5′-TGT CGT CCC AGT TGG TAA CA-3′ (103-bp product). The amplification and detection of the cDNA was accomplished using a StepOne Real-Time PCR System (Applied Biosystems) with 2 × QuantiTect SYBR Green PCR Master Mix (Qiagen) in a total volume of 20 µL.

Western blots

RAW 264 cells (5 × 105) were pretreated for 16 h with gAd in 24-well plates. Culture supernatants were removed and the cells were stimulated with A. actinomycetemcomitans lipopolysaccharide (100 ng mL−1) for 30 min. Cell extracts were prepared and resolved by 7% or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis before being transferred to polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA). After blocking, the membranes were incubated with primary and secondary antibodies, then washed thoroughly and examined using ECL Plus (GE Healthcare). The densities of the bands were scanned and quantified using LAS-1000plus (FujiFilm, Tokyo, Japan). The primary antibodies included an anti-p38 mitogen-activated protein kinase (MAPK) phospho-specific antibody, anti-c-Jun N-terminal kinase (JNK) phospho-specific antibody, anti-IκB kinase (IKK) α/β phospho-specific antibody, anti-IKKβ antibody, anti-IκB antibody (Cell Signaling Technology, Danvers, MA), antiextracellular signal-regulated kinase (ERK) 1/2 phospho-specific antibody (BioSource International, Camarillo, CA), anti-p38-MAPK antibody (BioLegend, San Diego, CA), anti-JNK antibody (R&D Systems, Minneapolis, MN), anti-ERK1/2 antibody (Abcam, Cambridge, MA), and anti-actin antibody (MP Biomedicals, Solon, OH). HRP-linked anti-rabbit immunoglobulin G (IgG) antibodies (Cell Signaling Technology) and HRP-linked anti-mouse IgG antibodies (Zymed Laboratories, South San Francisco, CA) were used as secondary antibodies.

Determination of TNF-α, IL-10, and IL-1β concentrations

RAW 264 cells (5 × 105) were pretreated for 16 h with gAd in 24-well plates. Culture supernatants were removed and the cells were stimulated with A. actinomycetemcomitans lipopolysaccharide (100 ng mL−1) for 12 h. Commercial enzyme-linked immunosorbent assay kits (Pierce Biotechnology, Rockford, IL) were used according to the manufacturer's protocols to quantify TNF-α, IL-10, and IL-1β concentrations in the cell-free culture supernatants.

Statistical analysis

Statistical analysis was performed using Student's t-test, and P<0.05 was considered significant.

Results

Effect of gAd on A. actinomycetemcomitans lipopolysaccharide-induced p38, JNK, ERK, IKK, and IκB phosphorylation and the degradation of IκB

RAW 264 cells were pretreated with 2 µg mL−1 gAd for 16 h. Culture supernatants were removed and the cells were stimulated in a fresh medium containing 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 30 min. Results from the Western blot showed that the pretreatment with 2 µg mL−1 gAd clearly inhibited the A. actinomycetemcomitans lipopolysaccharide-stimulated phosphorylation of p38-MAPK, JNK, ERK, and IKKα/β (Fig. 1). To assess the effect of gAd pretreatment on nuclear factor-κB activation, we monitored the degradation of IκB in response to lipopolysaccharide. Pretreatment with 2 µg mL−1 gAd partially reduced the A. actinomycetemcomitans lipopolysaccharide-induced degradation of IκB in RAW 264 cells (Fig. 1).

Figure 1

gAd-attenuated Aggregatibacter actinomycetemcomitans lipopolysaccharide (A. a. LPS)-induced phosphorylation of p38-MAPK, JNK, ERK, and IKK and degradation of IκB signaling pathways. RAW 264 cells pretreated with 2 µg mL−1 gAd for 16 h. Culture supernatants were removed and the cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 30 min. A single representative experiment (of three) is shown.

Figure 1

gAd-attenuated Aggregatibacter actinomycetemcomitans lipopolysaccharide (A. a. LPS)-induced phosphorylation of p38-MAPK, JNK, ERK, and IKK and degradation of IκB signaling pathways. RAW 264 cells pretreated with 2 µg mL−1 gAd for 16 h. Culture supernatants were removed and the cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 30 min. A single representative experiment (of three) is shown.

Effect of gAd on A. actinomycetemcomitans lipopolysaccharide-induced TNF-α mRNA expression and protein production

RAW 264 cells were pretreated with different concentrations of gAd (0.5–2 µg mL−1) for 16 h. The culture supernatants were removed and the cells were stimulated in a fresh medium containing 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide. Pretreatment with gAd reduced the A. actinomycetemcomitans lipopolysaccharide-induced TNF-α mRNA expression (Fig. 2a) and TNF-α protein production (Fig. 2b). Pretreatment with 2 µg mL−1 gAd reduced the A. actinomycetemcomitans lipopolysaccharide-induced TNF-α mRNA expression by 43.7±14.7% (Fig. 2a). As shown in Fig. 2b, the A. actinomycetemcomitans lipopolysaccharide-induced TNF-α protein was reduced markedly by pretreatment with 2 µg mL−1 gAd (3.36±0.32 ng mL−1) compared with pretreatment with medium alone (17.81±1.06 ng mL−1).

Figure 2

gAd-suppressed Aggregatibacter actinomycetemcomitans lipopolysaccharide (A. a. LPS)-induced TNF-α production. (a) RAW 264 cells pretreated with gAd for 16 h. Culture supernatants were removed and the cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 2 h. TNF-α mRNA expression was measured by real-time PCR. The values are presented as the mean ± SD, n=3. *P<0.05; **P<0.01 compared with cells stimulated with only A. actinomycetemcomitans lipopolysaccharide. (b) RAW 264 cells pretreated with gAd for 16 h. Culture supernatants were removed and the cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 12 h. The amount of TNF-α secreted into the medium was measured by enzyme-linked immunosorbent assay. Values are presented as the mean ± SD, n=3. *P<0.05; **P<0.01 compared with cells stimulated with only A. actinomycetemcomitans lipopolysaccharide.

Figure 2

gAd-suppressed Aggregatibacter actinomycetemcomitans lipopolysaccharide (A. a. LPS)-induced TNF-α production. (a) RAW 264 cells pretreated with gAd for 16 h. Culture supernatants were removed and the cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 2 h. TNF-α mRNA expression was measured by real-time PCR. The values are presented as the mean ± SD, n=3. *P<0.05; **P<0.01 compared with cells stimulated with only A. actinomycetemcomitans lipopolysaccharide. (b) RAW 264 cells pretreated with gAd for 16 h. Culture supernatants were removed and the cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 12 h. The amount of TNF-α secreted into the medium was measured by enzyme-linked immunosorbent assay. Values are presented as the mean ± SD, n=3. *P<0.05; **P<0.01 compared with cells stimulated with only A. actinomycetemcomitans lipopolysaccharide.

Effect of gAd on A. actinomycetemcomitans lipopolysaccharide-induced IL-1β mRNA expression

We next examined the effect of gAd pretreatment on A. actinomycetemcomitans lipopolysaccharide-induced IL-1β expression and secretion. Pretreatment with gAd reduced the A. actinomycetemcomitans lipopolysaccharide-induced IL-1β mRNA expression (Fig. 3). Pretreatment with 2 µg mL−1 gAd reduced the A. actinomycetemcomitans lipopolysaccharide-induced IL-1β mRNA expression by 79.1±1.7% (Fig. 3). Unexpectedly, we could not detect IL-1β protein in the culture medium of 3 µg mL−1A. actinomycetemcomitans lipopolysaccharide-treated RAW 264 cells (data not shown).

Figure 3

gAd-suppressed Aggregatibacter actinomycetemcomitans lipopolysaccharide (A. a. LPS)-induced IL-1β expression. RAW 264 cells pretreated with gAd for 16 h. Culture supernatants were removed and the cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 2 h. IL-1β mRNA expression was measured by real-time PCR. Values are presented as the mean ± SD, n=3. *P<0.05; **P<0.01 compared with cells stimulated with only A. actinomycetemcomitans lipopolysaccharide.

Figure 3

gAd-suppressed Aggregatibacter actinomycetemcomitans lipopolysaccharide (A. a. LPS)-induced IL-1β expression. RAW 264 cells pretreated with gAd for 16 h. Culture supernatants were removed and the cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 2 h. IL-1β mRNA expression was measured by real-time PCR. Values are presented as the mean ± SD, n=3. *P<0.05; **P<0.01 compared with cells stimulated with only A. actinomycetemcomitans lipopolysaccharide.

Effect of gAd on A. actinomycetemcomitans lipopolysaccharide-induced IL-10 mRNA expression and IL-10 protein production

Furthermore, we assessed the effect of gAd pretreatment on IL-10 expression and production (Fig. 4). Pretreatment with 0.5 µg mL−1 gAd augmented the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 mRNA expression by 2.59±0.37-fold (Fig. 4a), while pretreatment with 1 and 2 µg mL−1 gAd slightly increased the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 mRNA expression (Fig. 4a). As shown in Fig. 4b, the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 protein was enhanced by pretreatment with 0.5 µg mL−1 gAd (791.7±19.0 pg mL−1), 1 µg mL−1 gAd (699.5±67.6 pg mL−1), and 2 µg mL−1 gAd (877.8±39.5 pg mL−1) compared with pretreatment with medium alone (257.2±23.6 pg mL−1).

Figure 4

gAd-enhanced Aggregatibacter actinomycetemcomitans lipopolysaccharide (A. a. LPS)-induced IL-10 production. (a) RAW 264 cells pretreated with gAd for 16 h. Culture supernatants were removed and cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 2 h. IL-10 mRNA expression was measured by real-time PCR. Values are presented as the mean ± SD, n=3. *P<0.05; **P<0.01 compared with cells stimulated with only A. actinomycetemcomitans lipopolysaccharide. (b) RAW 264 cells pretreated with gAd for 16 h. Culture supernatants were removed and the cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 12 h. The amount of IL-10 secreted into the medium was measured by ELISA. Values are presented as the mean ± SD, n=3. *P<0.05; **P<0.01 compared with cells stimulated with only A. actinomycetemcomitans lipopolysaccharide.

Figure 4

gAd-enhanced Aggregatibacter actinomycetemcomitans lipopolysaccharide (A. a. LPS)-induced IL-10 production. (a) RAW 264 cells pretreated with gAd for 16 h. Culture supernatants were removed and cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 2 h. IL-10 mRNA expression was measured by real-time PCR. Values are presented as the mean ± SD, n=3. *P<0.05; **P<0.01 compared with cells stimulated with only A. actinomycetemcomitans lipopolysaccharide. (b) RAW 264 cells pretreated with gAd for 16 h. Culture supernatants were removed and the cells were stimulated with 100 ng mL−1A. actinomycetemcomitans lipopolysaccharide for 12 h. The amount of IL-10 secreted into the medium was measured by ELISA. Values are presented as the mean ± SD, n=3. *P<0.05; **P<0.01 compared with cells stimulated with only A. actinomycetemcomitans lipopolysaccharide.

Discussion

In the present study, we demonstrated that pretreatment with gAd reduced the A. actinomycetemcomitans lipopolysaccharide-induced phosphorylation of p38-MAPK, JNK, ERK, and IKK, the degradation of IκB, TNF-α production, and IL-1β mRNA expression in RAW 264 cells. Moreover, we found that pretreatment with gAd enhanced the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 production in RAW 264 cells.

Monocytes and macrophages play a central role in the inflammatory reactions caused by pathogenic bacteria (Kato et al., 2005). Moreover, they are both present in higher numbers in active periodontal lesions than in inactive sites (Zappa et al., 1991) and are key members of the innate immune system.

The A. actinomycetemcomitans lipopolysaccharide has the ability to activate ERK, JNK, p38-MAPK, and IκB in the human gingival fibroblasts (Mochizuki et al., 2004). We demonstrated that pretreatment with gAd inhibits the A. actinomycetemcomitans lipopolysaccharide-induced phosphorylation of p38-MAPK, JNK, ERK, and IKK and the degradation of IκB (Fig. 1). According to a recent review (Patil & Kirkwood, 2007), p38-MAPK inhibitors may have therapeutic value for the inhibition of cytokine-driven inflammatory bone loss in periodontal inflammation. Based on these data, adiponectin was suggested to attenuate the pathogenic property of lipopolysaccharide by inhibiting p38-MAPK, JNK, and ERK signaling.

Adiponectin dampens the early phases of macrophage inflammatory responses, acting to inhibit the growth of myelomonocytic progenitor cells and decrease the ability of mature macrophages to respond to activation (Huang et al., 2008). In this study, we demonstrated that gAd pretreatment reduced the A. actinomycetemcomitans lipopolysaccharide-induced TNF-α production (Fig. 2). The levels of TNF-α and IL-1β induced by A. actinomycetemcomitans and Escherichia coli lipopolysaccharide are higher than those induced by Porphyromonas gingivalis lipopolysaccharide in human polymorphonuclear leukocytes (Yoshimura et al., 1997). Treatment of RAW 264.7 cells with adiponectin decreased E. coli lipopolysaccharide-stimulated TNF-α production (Park et al., 2007). TNF-α released by the A. actinomycetemcomitans-infected THP-1 cells resulted in elevated p38-MAPK activity (Kato et al., 2005). Recently, we reported that gAd attenuated A. actinomycetemcomitans lipopolysaccharide-induced inducible nitric oxide synthase mRNA expression and nitric oxide production (Yamaguchi et al., 2007). TNF-α and IL-1β show induction of osteoclast recruitment and/or differentiation, as well as bone-resorbing activity in periodontitis (Boch et al., 2001). Taken together, the plasma adiponectin would regulate the excessive inflammatory response to A. actinomycetemcomitans infection in periodontal lesions.

Pretreatment of gAd with proteinase K resulted in a gAd preparation, which did not affect A. actinomycetemcomitans lipopolysaccharide-induced TNF-α mRNA (data not shown). Thus, the effect was not attributable to contamination of recombinant gAd by lipopolysaccharide.

In primary human macrophages, pretreatment with adiponectin slightly increased the lipopolysaccharide-induced IL-10 production (Wolf et al., 2004). Our data showed that pretreatment with gAd substantially increased the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 protein production (Fig. 4b). IL-10, ‘an antagonist cytokine,’ has been reported to exert anti-inflammatory effects by attenuating proinflammatory cytokine formation (Howard & O'Garra, 1992). The upstream signal transduction pathway regulating IL-10 transcription is not completely understood. In a recent study, lipopolysaccharide-induced IL-10 gene expression was mediated by p38-MAPK, ERK, and JNK in RAW 264.7 cells (Liu et al., 2006). Moreover, cAMP-dependent signaling is linked to stress-induced IL-10 expression through cAMP-response element binding protein (CREB) (Platzer et al., 2000). The gAd increased the phosphorylation of CREB (Park et al., 2008), p38-MAPK, ERK, and JNK(Kamio et al., 2008). Additionally, gAd-induced IL-10 is mediated by CREB (Park et al., 2008). In the present study, however, the results of the experiments using inhibitors for PKC, cAMP, and NOS suggested that these molecules were not involved in the gAd signaling to enhance the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 production (data not shown). The signal pathway to enhance the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 production by gAd remains to be elucidated.

In conclusion, we demonstrated the anti-inflammatory activity of gAd by the inhibition of p38-MAPK, JNK, ERK and IKK phosphorylation, degradation of IκB, suppressing TNF-α production, and enhancement of IL-10 production in A. actinomycetemcomitans lipopolysaccharide-stimulated RAW 264 cells.

Acknowledgements

The authors thank Drs I. Shimomura and N. Maeda for generously providing the mouse gAd cDNA. Support for this research was provided by a Grant-in-Aid for Scientific Research (C) 18592285 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Author notes

Editor: Artur Ulmer