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The Journal of Immunology, 2006, 177: 3799-3805.
Copyright © 2006 by The American Association of Immunologists, Inc.

Receptor Activator of NF-{kappa}B Ligand and Osteoprotegerin Regulate Proinflammatory Cytokine Production in Mice1

Kenta Maruyama*, Yasunari Takada*, Neelanjan Ray*, Yukiko Kishimoto{dagger}, Josef M. Penninger{ddagger}, Hisataka Yasuda{dagger} and Koichi Matsuo2,*

* Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo, Japan; {dagger} Nagahama Institute for Biochemical Science, Oriental Yeast, Shiga, Japan; and {ddagger} Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Receptor activator of NF-{kappa}B ligand (RANKL) is a membrane-bound or soluble cytokine essential for osteoclast differentiation, whereas the decoy receptor osteoprotegerin (OPG) masks RANKL activity. In mouse serum, both soluble RANKL and OPG are detectable. We observed that mice injected with LPS showed significantly down-regulated serum RANKL levels, whereas serum OPG levels were up-regulated. However, the roles of RANKL and OPG in innate immunity remain obscure. We found that RANKL pretreatment suppressed production of proinflammatory cytokines in macrophages in response to stimulation by bacteria and their components. Furthermore, such RANKL-induced tolerance in macrophages was inhibited by GM-CSF treatment, which blocks RANKL signaling. RANKL-induced tolerance occurred in the absence of c-Fos, which is essential for osteoclast differentiation. In mice lacking OPG, LPS-induced production of proinflammatory cytokines was reduced, whereas in mice lacking RANKL, it was increased, and lethality following LPS injection was also elevated, suggesting that constitutive activities of RANKL suppress cytokine responsiveness to LPS in vivo. Strikingly, prior administration of RANKL protected mice from LPS-induced death. These data reveal prophylactic potential of RANKL in acute inflammatory diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Receptor activator of NF-{kappa}B ligand (RANKL,3 also known as osteoclast differentiation factor or TNF-related activation-induced cytokine) is a member of the TNF superfamily and is encoded by Tnfsf11 (1, 2, 3). Both membrane-bound and soluble forms of RANKL are produced by bone-forming osteoblasts. The signaling receptor for RANKL is called RANK (encoded by Tnfrsf11a) and is expressed in cells in the macrophage-osteoclast lineage. The decoy receptor osteoprotegerin (OPG, also called osteoclastogenesis inhibitory factor), which is encoded by Tnfrsf11b, masks the activity of RANKL (2, 4, 5). RANKL-RANK signaling induces differentiation of osteoclasts, which resorb bone. RANK recruits TNFR-associated factor (TRAF) adaptor molecules and eventually activates dimeric transcription factors such as AP-1 and NF-{kappa}B. Transcription factor c-Fos, an AP-1 subunit, is required for transcriptional activation of the osteoclastogenic transcription factor NFATc1 (6). Mice lacking RANKL (7), RANK (8, 9), TRAF6 (10, 11), NF-{kappa}B (p50 and p52) (12), c-Fos (13, 14, 15), or NFATc1 (16) develop osteopetrosis due to a differential block in the osteoclast lineage. Conversely, mice lacking OPG become osteopenic due to enhanced osteoclastogenesis (17, 18). Although much is known about how RANKL and OPG function in bone biology, their roles in the immune system are still obscure.

In rheumatoid arthritis patients, both activated T cells and synovial cells in inflammatory joints express membrane-bound and soluble RANKL, thereby promoting osteoclastogenesis and bone resorption. In contrast, RANKL promotes survival and supports function of dendritic cells (2, 19). Mice lacking RANKL show defects in early differentiation of T and B lymphocytes as well as lymph node organogenesis (7).

LPS, or endotoxin, is a cell-wall component of Gram-negative bacteria. High doses of LPS can result in production of excess amounts of proinflammatory cytokines, or a "cytokine storm," leading to endotoxin shock. Such a cytokine storm is a distinctive feature of systemic inflammatory response syndrome in humans, which is a leading cause of death in intensive care units; the incidence of sepsis as well as the number of sepsis-related deaths is on the rise (20). LPS stimulation of the TLR4 activates two signaling pathways: MyD88-dependent and MyD88-independent pathways. TLR4 signaling activates NF-{kappa}B, AP-1, and other transcription factors, which bind to promoters and enhancers of proinflammatory cytokine genes (21, 22). LPS tolerance, or suppression of immunological responses after exposure to LPS, is thought to protect animals from pathological hyperactivation of the innate immune response during infection. In this study, we show that RANKL, like LPS, can suppress proinflammatory cytokine responses to LPS and other bacterial components both in vitro and in vivo. RANKL may be useful for prophylactic treatment for acute excessive inflammatory responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Six- to 10-wk-old C57BL/6J mice were purchased from Oriental Yeast. Homozygous mice lacking OPG (Tnfrsf11b–/–) (18) and control heterozygotes on a C57BL/6 background were purchased from Clea Japan. Mice lacking RANKL (Tnfsf11–/–) (7) on a C57BL/6 background and Fos–/– mice (13) on a 129 x C57BL/6 mixed background were bred and maintained under specific pathogen-free conditions. A powder diet was provided to Fos–/– and Tnfsf11–/– mice. All experiments were performed in accordance with guidelines for animal use at the Keio University School of Medicine.

M-CSF-dependent macrophages

To generate M-CSF-dependent bone marrow-derived macrophages (MDBMs), bone marrow cells were harvested by flushing tibias and femurs with DMEM containing 10% FCS and antibiotics. After passage through a cell strainer, bone marrow cells were cultured overnight. Nonadherent cells were harvested and cultured in the presence of 10 ng/ml M-CSF (R&D Systems). After 3–4 days, adherent cells were collected using a cell scraper, and seeded as MDBMs at 1 x 105/well in a 24-well plate (Falcon). M-CSF-dependent spleen-derived macrophages (MDSMs) were generated similarly from splenocytes. All macrophages were cultured in the presence of 10 ng/ml M-CSF in all experiments.

Peritoneal macrophages

Peritoneal cells were harvested by flushing the peritoneal cavity of mice with complete medium. A total of 1 x 105 cells/well were seeded in 24-well plates (Falcon). Cells were cultured for 6 h, washed with PBS to remove nonadherent cells, and incubated in fresh medium. These adherent cells were used as peritoneal macrophages.

In vitro tolerance experiments

Macrophages were pretreated with different concentrations of RANKL (<1 endotoxin units (EU)/µg; R&D Systems) or LPS (Salmonella minnesota Re595; Sigma-Aldrich) for the indicated periods of time. Cultures were subsequently washed twice with PBS and stimulated with LPS, flagellin from Salmonella muenchen (Calbiochem), or phosphorothioate CpG (5'-TCCATgACgTTCCTgATgCT-3') or control GpC (5'-TCCATgAgCTTCCTgATgCT-3') oligonucleotides (Proligo) as indicated. In some experiments, 500 U/ml GM-CSF (PeproTech) was added for 3 h before cells were stimulated with LPS.

LPS and RANKL injection in mice

LPS (S. minnesota Re595; Sigma-Aldrich) was administered i.p. Blood was collected by heart puncture at the indicated time points. Blood was allowed to clot for 1 h and then centrifuged at 15,000 rpm for 20 min at 24°C. The serum was stored at –80°C until cytokine assays. A mixture of 10 µg of RANKL (GST-RANKL; Oriental Yeast; unpublished observations) and 2 µg of M-CSF (Leukoprol; Kyowa Hakko) in PBS, or PBS alone was injected i.p. 24 h before 2.5 mg/mouse LPS (Escherichia coli serotype O55:B5; Sigma-Aldrich) administration.

Bacterial strain and infection

For infection, an overnight standing culture of Salmonella enterica serovar Typhimurium strain {chi}3306 (23) in Luria-Bertani broth was diluted and shaken, and mid-log phase bacteria were then collected by centrifugation. Bacteria were washed with PBS, diluted with Hanks’ salt solution, and used to infect macrophages at a multiplicity of infection of 10. Cultures were incubated at 37°C for 1 h, washed with PBS to remove extracellular bacteria, and then incubated in complete medium containing 25 µg/ml gentamicin. After 3 h, culture supernatants were harvested for cytokine assays. For oral infection, mice were fasted without water for 12 h and then 3.3 x 107 CFU/g body weight of Salmonella were administered orally. Four days later, blood was collected by heart puncture.

ELISA

TNF-{alpha}, IL-6, and IL-12 p40 levels in macrophage culture supernatants were measured using ELISA sets (BD Pharmingen). RANKL and OPG levels in mouse serum were measured using ELISA kits (R&D Systems).

Western blot analysis

Western blotting was performed as described (24). The following primary Abs were used: polyclonal anti-MD-2 rabbit Ab (FL-160; Santa Cruz Biotechnology) and a polyclonal anti-TLR4 goat Ab (L-14; Santa Cruz Biotechnology). Blots were stripped and reprobed with a polyclonal anti-actin goat Ab (Santa Cruz Biotechnology) to monitor protein loading.

RT-PCR analysis

Macrophages were collected and homogenized in Isogen (Nippon Gene) for purification of RNA. cDNA was synthesized using the Enhanced Avian HS RT-PCR kit (Sigma-Aldrich). Quantitative PCR was performed on an ABI PRISM 7000 using TaqMan Assay-on-demand (Applied Biosystems) for IL-6, TNF-{alpha}, IL-12 p40, c-Fos (Fos), CD14 (Cd14), TLR4 (Tlr4), MD2 (Ly96), TRIF (Ticam1), TRAM (Ticam2), IL-1R-associated kinase 4 (Irak4), MyD88 (Myd88), and Gapdh.

Statistical analysis

Data are expressed as means ± SD. All data were analyzed using the Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
RANKL induces tolerance to bacterial components in macrophages

We first compared proinflammatory cytokine production by MDBMs treated with either LPS or RANKL (Fig. 1). While LPS induced production of significant amounts of TNF-{alpha}, IL-6, and IL-12 p40, RANKL, even as high as 100 ng/ml, did not induce detectable levels of cytokines. Therefore, unlike LPS, RANKL cannot induce proinflammatory cytokines.


Figure 1
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  		FIGURE 1. Lack of cytokine induction by RANKL. MDBMs were treated with LPS or RANKL at indicated concentrations for 24 h. Protein levels of proinflammatory cytokines in culture supernatants were measured by ELISA. Bars represent means ± SD (n = 3 culture wells).

 
We treated MDBMs first with increasing concentrations of RANKL for 24 h, and then with LPS for 6 h. Levels of TNF-{alpha}, IL-6, and IL-12 p40 in supernatants were measured by ELISA. RANKL pretreatment inhibited LPS-induced production of these cytokines in a dose-dependent manner (Fig. 2A). RANKL-induced tolerance was observed at RANKL concentrations as low as 1 ng/ml. We also examined expression of TNF-{alpha}, IL-6, and IL-12 p40 mRNA over 6 h after LPS stimulation. Compared with nonpretreated macrophages, RANKL-pretreated macrophages showed significantly reduced expression of cytokine mRNAs upon LPS stimulation (Fig. 2B). The duration of RANKL pretreatment was shortened from 24 h to 12 or 6 h; pretreatment for as short as 6 h significantly suppressed LPS-induced TNF-{alpha} production (Fig. 2C). To determine whether RANKL induces tolerance to LPS in other macrophages than MDBMs, we assayed peritoneal macrophages and M-CSF-dependent spleen-derived macrophages (MDSMs). These macrophages also showed suppressed production of TNF-{alpha} in response to LPS when pretreated with RANKL (Fig. 2D). These data demonstrate that RANKL renders macrophages tolerant to LPS.


Figure 2
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  		FIGURE 2. RANKL-induced tolerance in macrophages. A, Protein levels of proinflammatory cytokines. MDBMs were pretreated with RANKL for 24 h (1°) and then stimulated with 100 ng/ml LPS for 6 h (2°). B, mRNA levels. MDBMs were pretreated with medium alone ({diamond}) or with 10 ng/ml RANKL ({diamondsuit}) for 24 h and then stimulated with 100 ng/ml LPS for 6 h. Cells were harvested at the indicated time points, and cytokine levels were measured by quantitative RT-PCR. Values are normalized to Gapdh. C, MDBMs were pretreated with 1 ng/ml RANKL for the indicated period of time (1°) and stimulated with LPS (2°). D, Peritoneal macrophages and MDSMs were pretreated with 50 ng/ml RANKL for 24 h (1°) and then stimulated with LPS as in (2°). *, p < 0.05; **, p < 0.01 vs controls (no RANKL).

 
Attenuation of tolerance by RANKL removal or GM-CSF treatment

To determine whether RANKL-induced tolerance is reversible, we treated MDBMs with RANKL for 24 h, and then with RANKL-free medium for another 24 h. Compared with controls, production of TNF-{alpha}, IL-6, and IL-12 p40 in response to LPS was fully or at least partially restored in these cells, suggesting that RANKL-induced tolerance is reversible (Fig. 3A). Because GM-CSF can interfere with RANKL/RANK signaling (25), we next asked whether GM-CSF affects RANKL-induced tolerance. MDBMs were pretreated with RANKL for 24 h and then treated with GM-CSF for 3 h in the absence of RANKL. Cells were then stimulated with LPS, and TNF-{alpha} levels were determined (Fig. 3B). While GM-CSF did not induce TNF-{alpha} production by itself, the short exposure to GM-CSF efficiently increased TNF-{alpha} production in RANKL-pretreated macrophages, indicating that GM-CSF attenuates RANKL-induced tolerance (Fig. 3B). Taken together, these data suggest that removal of RANKL, as well as brief treatment with GM-CSF, can restore responsiveness of macrophages to LPS.


Figure 3
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  		FIGURE 3. Attenuation of RANKL-induced tolerance. A, MDBMs were treated with medium alone (Med) or 10 ng/ml RANKL for 24 h (day 1), stimulated with medium alone (Med), or with 10 ng/ml RANKL for another 24 h (day 2), and then stimulated with 100 ng/ml LPS for 6 h. Supernatants (n = 3) were harvested and cytokine levels were measured by ELISA. Bars represent means ± SD. *, p < 0.01 vs {blacksquare}. B, MDBMs were pretreated with 10 ng/ml RANKL for 24 h (1°), then stimulated with 500 U/ml GM-CSF for 3 h (2°), and then stimulated with 100 ng/ml LPS for 6 h (3°). TNF-{alpha} levels in supernatants (n = 3) were determined by ELISA. Bars represent means ± SD. *, p < 0.01. Experiments shown are representative of three assays.

 
RANKL-induced tolerance is c-Fos independent

We next examined whether c-Fos, which mediates RANKL signaling during osteoclast differentiation, is involved in LPS- or RANKL-induced tolerance. We prepared MDSMs from mice lacking c-Fos, and first pretreated these cells with a low dose of LPS for 24 h. Fos–/– macrophages show enhanced production of TNF-{alpha}, compared with wild-type controls as previously reported (26). However, regardless of the genotype, LPS pretreatment resulted in suppressed production of TNF-{alpha}, and IL-6 by >80% in response to the second stimulation, compared with nonpretreated controls, suggesting that c-Fos is dispensable for LPS-induced tolerance (Fig. 4, upper). We next determined whether RANKL-induced tolerance occurs in the absence of c-Fos. RANKL pretreatment of Fos–/– macrophages significantly suppressed production of TNF-{alpha} and IL-6 in a dose-dependent manner (Fig. 4, lower). These data suggest that not only LPS- but also RANKL-induced tolerance is c-Fos independent.


Figure 4
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  		FIGURE 4. LPS- or RANKL-induced tolerance in Fos–/– macrophages. Upper panels, LPS-induced tolerance. MDBMs from wild-type ({blacksquare}) and MDSMs from Fos–/– mice ({square}) were pretreated with 1 ng/ml LPS for 24 h (1°), and then stimulated with 100 ng/ml LPS for 6 h (2°). Cytokine levels were determined in supernatants by ELISA. Bars represent means ± SD (n = 3). *, p < 0.05; **, p < 0.01 vs no LPS pretreatment. Lower panels, RANKL-induced tolerance. MDSMs from Fos –/– mice were pretreated with RANKL at the indicated concentrations for 24 h (1°), and then stimulated with 100 ng/ml LPS for 6 h (2°). Supernatant cytokine levels (n = 3 culture wells) were determined by ELISA. Bars represent means ± SD. *, p < 0.05; **, p < 0.01; compared with no RANKL pretreatment.

 
RANKL treatment reduces MyD88 expression

To further gain insight into the molecular mechanisms underlying RANKL-induced tolerance, we measured expression levels of TLR4 and MD2 in MDBMs after RANKL treatment. By Western blotting, there were no significant changes in levels of these molecules (Fig. 5A). We next examined mRNA levels of molecules associated with the TLR4-signaling pathway over 24 h after RANKL treatment. In contrast to c-Fos (Fos) mRNA, which was increased nearly 4-fold, MyD88 (Myd88) mRNA was significantly reduced (Fig. 5B). Other molecules such as CD14 (Cd14), TLR4 (Tlr4), MD2 (Ly96), TRIF (Ticam1), TRAM (Ticam2), and IRAK4 (Irak4) showed little or only transient changes (Fig. 5B). Since MyD88 is a common adaptor molecule for multiple TLRs which recognize distinct bacterial components, we stimulated MDBMs with flagellin, CpG oligonucleotides, and Salmonella after pretreatment with RANKL for 24 h. RANKL pretreatment suppressed TNF-{alpha} production in response to the bacterial components and Salmonella (Fig. 6). These data are consistent with the notion that down-regulation of MyD88 may be a mechanism for RANKL-induced tolerance in response to various bacterial components.


Figure 5
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  		FIGURE 5. Expression of TLR4-signaling molecules after RANKL treatment. A, Western blot analysis. Wild-type MDBMs were treated with indicated concentrations of RANKL for 24 h. B, Quantitative RT-PCR analysis. Wild-type MDBMs were treated with 10 ng/ml RANKL for indicated hours. mRNA levels were measured for c-Fos (Fos), CD14 (Cd14), TLR4 (Tlr4), MD2 (Ly96), TRIF (Ticam1), TRAM (Ticam2), IRAK4 (Irak4), MyD88 (Myd88), and normalized with Gapdh. Bars represent means ± SD.

 

Figure 6
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  		FIGURE 6. RANKL-induced tolerance to various stimulants. MDBMs were pretreated with 10 ng/ml RANKL for 24 h (1°) and stimulated with 1 x 10–11M flagellin or 3 nM CpG DNA for 6 h, and Salmonella for 3 h (2°). Cytokine levels were determined in culture supernatants by ELISA. Bars represent means ± SD (n = 3 wells). *, p < 0.05; **, p < 0.01 vs controls ({square}).

 
Serum RANKL and OPG levels shift dynamically upon LPS and bacterial challenge

To compare physiological and pathological RANKL concentrations in serum, we injected LPS i.p. into mice and then quantified serum RANKL and OPG levels. The RANKL concentration was ~150 pg/ml and the OPG concentration was ~2000 pg/ml in serum before LPS injection. Surprisingly, RANKL concentration dramatically fell 3–6 h after LPS injection, while OPG concentration was up-regulated 12–24 h after LPS injection (Fig. 7A). Down-regulation of RANKL and induction of OPG were also observed after Salmonella oral infection on day 4 (Fig. 7B). These data revealed that RANKL and OPG levels are dynamically regulated in response to endotoxin and bacterial infection.


Figure 7
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  		FIGURE 7. Serum RANKL and OPG levels. A, LPS challenge. C57BL/6J mice (n = 24) were i.p. injected with LPS (1 µg/g body weight). Blood was collected at the indicated time points (n = 3 mice/point). Serum RANKL and OPG were quantified by ELISA. Bars represent means ± SD. *, p < 0.05; **, p < 0.01 vs 0 h. B, Salmonella challenge. C57BL/6J mice (n = 6) were inoculated orally with 3.3 x 107 CFU/g body weight of Salmonella (infected) or PBS (control). After 4 days, blood was collected and serum RANKL and OPG were quantified by ELISA. Bars represent means ± SD (n = 3). *, p < 0.05; **, p < 0.01.

 
Hypersensitivity of RANKL-deficient mice to endotoxin

To determine whether constitutive levels of serum RANKL influence LPS-induced cytokine production in mice, we analyzed Tnfsf11–/– mice, which lack RANKL. As expected, serum RANKL was not detectable in Tnfsf11–/– mice, whereas OPG levels were slightly higher in Tnfsf11–/– mice than in wild-type controls (Fig. 8A). After LPS injection, we observed significantly elevated production of TNF-{alpha} and IL-6 in Tnfsf11–/– mice (Fig. 8B). These data are consistent with the notion that the lack of RANKL-induced tolerance potentiates production of proinflammatory cytokines in Tnfsf11–/– mice.


Figure 8
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  		FIGURE 8. Proinflammatory cytokine production in mice lacking RANKL or OPG. A, Serum RANKL and OPG levels in mice lacking RANKL (Tnfsf11–/–) and wild-type control mice (n = 4 for each genotype) were measured by ELISA. B, Serum cytokine levels 90 min after i.p. injection of 2 µg/g LPS into Tnfsf11–/– and wild-type mice. C, Serum RANKL and OPG levels in mice lacking OPG (Tnfrsf11b–/–) and heterozygous control mice (n = 5 for each genotype) were measured by ELISA. D, Serum cytokine levels 90 min after i.p. injection of 2 µg/g LPS into Tnfrsf11b–/– and heterozygous control mice, cytokine levels were measured by ELISA. Bars represent means ± SD. *, p < 0.05; **, p < 0.01 vs controls. E, Hypersensitivity of Tnfsf11–/– mice to LPS. Survival curves were generated after injecting a high dose of LPS (130 µg/g) LPS i.p. into 6-wk-old wild-type (n = 10) or Tnfsf11–/– mice (n = 5).

 
To further examine the role of RANKL-induced tolerance in mice, we analyzed Tnfrsf11b–/– mice, which lack OPG. In comparison to control heterozygotes, constitutive serum RANKL levels were ~10-fold higher in Tnfrsf11b–/– (Fig. 8C). Therefore, mice lacking OPG are constitutively exposed to higher RANKL levels. On LPS injection, we observed significantly decreased production of IL-6 in Tnfrsf11b–/– mice than in heterozygous controls (Fig. 8D). Therefore, chronic exposure to high RANKL levels attenuates the proinflammatory cytokine response in mice lacking OPG.

To analyze the effect of dysregulated production of proinflammatory cytokines in mice lacking RANKL, we injected a high dose of LPS i.p. into wild-type and Tnfsf11–/– mice. While 90% of wild-type control mice (n = 10) survived 24 h postinfection, every Tnfsf11–/– mouse (n = 5) died within 24 h (Fig. 8E). These data suggest that constitutive levels of serum RANKL protect against endotoxin shock in mice.

RANKL injection protects mice from endotoxic shock

To explore therapeutic potential of RANKL against endotoxic shock, we injected into mice a mixture of RANKL and M-CSF i.p. 24 h before administration of a lethal dose of LPS, which killed 90% of control mice within 80 h. Strikingly, when pretreated with RANKL and M-CSF, over 60% of mice survived (Fig. 9). These data suggest that RANKL can be used as a prophylactic molecule to prevent endotoxic shock.


Figure 9
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  		FIGURE 9. RANKL pretreatment protects mice from LPS-induced death. A mixture of 10 µg of GST-RANKL and 2 µg of M-CSF in PBS (RANKL, {square}) or PBS alone (PBS, {blacksquare}) was injected i.p. 24 h before 2.5 mg/mouse LPS administration into C57BL6/J mice (n = 10 each group, 7–8 wk old), then mice were observed for 144 h to generate survival curves.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we demonstrate that RANKL stabilizes the innate immune system challenged by endotoxin. Excessive production of proinflammatory cytokines in response to microbial components has been shown to be negatively regulated by multiple mechanisms in mice (27, 28, 29, 30). Our findings reveal that RANKL can also induce tolerance and suppress proinflammatory cytokine production in macrophages.

RANKL-induced tolerance is reversible, given that RANKL-pretreated cells regain responsiveness to LPS after incubation in RANKL-free medium. Furthermore, RANKL-tolerance occurred in various macrophages, including peritoneal macrophages as well as M-CSF dependent, bone marrow- or spleen-derived macrophages. Therefore, RANKL likely establishes tolerance in virtually all macrophages.

One may think that RANKL-induced tolerance could be due to osteoclast differentiation of these macrophages, since mature osteoclasts do not respond efficiently to LPS via TLR4 (31, 32). However, macrophages lacking c-Fos, which are deficient for osteoclastogenesis, also exhibit RANKL-induced tolerance. Therefore, RANKL-induced tolerance is likely to occur independently of osteoclast formation. Analyses of mRNA expression related to the TLR4-signaling pathway revealed that MyD88 expression is reduced by RANKL treatment. Since MyD88 is a core adaptor molecule for various TLRs, reduced MyD88 expression should also affect other TLR signaling. In fact, we observed that cytokine production in response to flagellin and CpG DNA is also suppressed by RANKL pretreatment. Therefore, down-regulation of MyD88 may be a molecular basis for RANKL-induced tolerance.

Upon LPS injection or Salmonella oral infection, we observed that serum RANKL levels were down-regulated within 5 h, while serum OPG levels were up-regulated over 24 h. Down-regulation of RANKL in response to LPS or infection was unexpected, because bacterial infection increases RANKL expression at both the mRNA and protein level in osteoblasts and synovial fibroblasts (33, 34). Therefore, down-regulation of serum RANKL could require posttranslational regulation, such as suppression of RANKL shedding (35, 36). We also showed that GM-CSF, which suppresses RANKL signaling (25), counteracts RANKL-induced tolerance. GM-CSF is also induced in serum by LPS injection into mice (37) and potentiates LPS-mediated release of TNF-{alpha}, IL-1, and IL-6 by macrophages. Consistently, mice lacking GM-CSF exhibit reduced production of proinflammatory cytokines in response to LPS and show increased resistance to endotoxin (38). Therefore, it appears that the immune system switches off RANKL-induced tolerance upon severe bacterial infection by inducing GM-CSF and OPG and down-regulating RANKL. In addition, these observations suggest, for the first time, that the RANKL-OPG ratio in serum is an indicator of endotoxemia and bacterial infection.

Constitutive levels of RANKL seem to induce tolerance in mice. In response to LPS injection, mice lacking RANKL showed increased production of proinflammatory cytokines, especially TNF-{alpha}. In contrast, mice lacking OPG showed decreased production of IL-6 in response to LPS. Moreover, peritoneal macrophages lacking OPG also showed reduced cytokine responses to LPS ex vivo (data not shown). To reveal significance of RANKL in suppressing endotoxin shock, we examined survival of mice lacking RANKL after LPS injection. RANKL-deficient mice were highly susceptible to LPS compared with wild-type controls, demonstrating that RANKL attenuates proinflammatory cytokine production in mice exposed to LPS. Moreover, pretreatment of mice with RANKL in combination with M-CSF protects mice from LPS-induced mortality. These data suggest that RANKL can be used as a preventive molecule against endotoxin shock.

Taken together, these results indicate that RANKL suppresses production of proinflammatory cytokines by macrophages in response to LPS and other bacterial components. We also showed that RANKL can be used as a prophylactic molecule to prevent endotoxic shock. Thus far, RANKL and OPG have been investigated mainly in the context of bone biology, but our data provide evidence that RANKL and OPG dynamically regulate cytokine responses in infectious diseases.


   Acknowledgments
 
We thank H. Ishikawa for discussion, and S. Koyasu and E. Lamar for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
K. Maruyama, H. Yasuda, and K. Matsuo, along with Keio University and Oriental Yeast, have a pending patent on a method of detecting, and a composition for preventing or treating, inflammatory diseases.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects, and in part by Done Science. Back

2 Address correspondence and reprint requests to Dr. Koichi Matsuo, Department of Microbiology and Immunology, School of Medicine, Keio University, 160-8582 Tokyo, Japan. E-mail address: matsuo{at}sc.itc.keio.ac.jp Back

3 Abbreviations used in this paper: RANKL, receptor activator of NF-{kappa}B ligand; OPG, osteoprotegerin; TRAF, TNFR-associated factor; MDBM, M-CSF-dependent bone marrow-derived macrophage; MDSM, M-CSF-dependent spleen-derived macrophage; TRIF, Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta; TRAM, TRIF-related adaptor molecule; IRAK, IL-1R-associated kinase. Back

Received for publication January 27, 2006. Accepted for publication June 29, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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