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. 2009 Jul 29;86(4):933–940. doi: 10.1189/jlb.0708419

Regulation of dendritic cell survival and cytokine production by osteoprotegerin

Takahiro Chino *,1, Kevin E Draves †, Edward A Clark †,‡
PMCID: PMC2752017  PMID: 19641036

Abstract

The TNF family ligand, RANKL, and its two TNFR family receptors, RANK and OPG, enable coordinated regulation between the skeletal and immune systems. Relatively little is known about how OPG influences RANKL-RANK interactions for the regulation of DCs. Here, we show that OPG KO bone marrow-derived DCs survive better and produce more TNF-α, IL-12p40, and IL-23 in response to Escherichia coli LPS than WT DCs. RANKL is induced on DCs within 24 h after LPS stimulation. OPG limits RANKL-RANK interactions between DCs, which can promote DC survival and elevated expression of proinflammatory cytokines. Survival of and cytokine production by OPG KO DCs are inhibited by soluble OPG; conversely, anti-OPG enhances survival and cytokine production by WT DCs. Bim KO DCs, like OPG KO, also survive longer and produce more TNF-α than WT DCs; however, unlike OPG KO, Bim KO DCs do not produce more IL-23. In addition, after inoculation with LPS, OPG KO mice produce more TNF-α and IL-12p40 than WT mice but not more IL-6. Thus, OPG regulates not only DC survival but also the nature of DC-dependent inflammatory responses.

Keywords: inflammation, anti-inflammation, lifespan

Introduction

DCs are very effective at recognizing, capturing, and processing antigens and after activation, are highly effective APCs [1]. DC also regulate inflammatory responses and play an important role in coordinating innate and adaptive immune responses against pathogens [2]. A key element in the initiation of an innate immune response against pathogens is the recognition of PAMPs, components found commonly on pathogens but not found normally on host cells [3]. DCs recognize PAMPs through pattern recognition receptors including TLRs and CLRs. Upon infection, DCs expressing TLRs and CLRs bind PAMPs and initiate signaling pathways that promote host defenses [4] LPS, a PAMP that interacts with TLR4, is a major component of gram-negative bacteria. It induces DCs to mature and to produce a proinflammatory cytokine such as IL-12. In addition to cytokine secretion, TLR signaling influences DC survival. Hou and Van Parijs [5] found that TLRs and T cell costimulatory molecules trigger a DC survival pathway that is dependent on Bcl-XL. However, TLRs uniquely increased expression of the proapoptotic BH3-only Bcl-2 family member, Bim [6], and promoted cell death.

DCs were initially considered end-stage, nondividing cells with a half-life between 1.5 and 2.9 days [7]. However, it is now known that DCs in peripheral lymphoid organs undergo a limited number of divisions and can survive up to 10–14 days [8]. The lifespan of activated DCs is enhanced by components of the innate and acquired immune systems. These include inflammatory cytokines and PAMPs that function as ligands for TLRs [9], as well as T cell-expressed costimulatory molecules, such as CD40 ligand [10] and RANKL [11]. Recent studies have suggested that the lifespan of DCs may affect quantity and quality of immune response [12,13,14]. Chen et al. [12] reported that targeted inhibition of DC apoptosis with a caspase inhibitor results in chronic lymphocyte activation and systemic autoimmune manifestations. The same group also found that Bim KO DCs have less spontaneous cell death than WT DCs and after adoptive transfer, efficiently induce T cell activation and autoantibody production [13].

The regulatory mechanisms controlling DC fate are mediated in part by DC interactions with T cells. Although the TNF family molecule RANKL and its receptor RANK are key regulators of bone remodeling and essential for osteoclastogenesis, RANKL also regulates DC survival [11, 15]. After RANKL on T cells binds to and signals through RANK on DCs, the NF-κB and JNK pathways are activated [16, 17]. Although RANKL does not induce up-regulation of costimulatory molecules, it does stimulate DCs to produce proinflammatory cytokines, including IL-12 [18, 19]. RANKL may enhance DC viability through activation of PI3K and by up-regulating Bcl-XL and the NF-κB pathway [9, 11, 20, 21]. Ouaaz et al. [21] found that p50/RelA double-KO mice have impaired DC but not macrophage development. They also found that p50/cRel double-KO DCs do not survive or make IL-12 and Bcl-2/Bcl-XL in response to RANKL or LPS. These results suggest that p50/cRel may play an important role in DC survival and IL-12 production.

In addition to DC-T cell interactions, interactions among DCs may generate survival signals. For example, human CD34+ iDCs express RANKL and RANK and are therefore capable of providing survival signals through DC-DC contact [22]. OPG, a soluble member of the TNFR family, functions as a decoy receptor for RANKL and thereby limits RANKL-RANK interactions. Accordingly, OPG KO mice develop osteoporosis [23, 24]. OPG is up-regulated by CD40 ligation on human DCs [25] and by LPS or estrogen [26]. OPG KO DCs are also hyperactive and more effective at stimulating allogeneic T cells than WT DCs [27].

In this study, we found that OPG KO DCs survive better than WT DCs and produce more TNF-α, IL-12p40, and IL-23 than WT DCs in response to Escherichia coli LPS. This is a result of sustained interactions between DCs, which increase the longevity of OPG KO DCs and the level of proinflammatory cytokines produced by OPG KO DCs. Conversely, OPG treatment reduced the survival of and cytokine production by WT DCs. Our results suggest that OPG regulates survival and cytokine production of DCs, thereby affecting the nature of inflammatory responses.

MATERIALS AND METHODS

Mice

C57BL/6 OPG KO mice, previously generated in our laboratory [27], were housed in the University of Washington Animal Care Facilities (Seattle, WA, USA) under specific pathogen-free conditions. We also used Bim KO mice (Bim KO B6.129-Bcl21lltm1.1Ast/J) and C57BL/6J mice, both of which were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). All procedures used were preapproved by the Institutional Animal Care and Use Committee.

Preparation of DCs

DCs were prepared from mouse bone marrow as described previously [27] with some modifications. Briefly, bone marrow cells from WT and OPG KO littermates were flushed from femurs and tibia. The harvested bone marrow cells were cultured in medium in the presence of GM-CSF (20 ng/ml, RDI, Concord, MA, USA). On Days 1 and 3 of the culture, the nonadherent cells were removed, and the remaining adherent cells were washed with RPMI 1640 and then fed with fresh medium containing GM-CSF every other day. These cells were used at Days 7–8 for experiments, at which time >90% were CD11c+ iDCs. In some experiments, cells at Days 7–8 were stained with anti-CD11c (PE-conjugated; eBioscience Inc., San Diego, CA, USA) and anti-Gr-1 (FITC-conjugated; BD Biosciences, San Jose, CA, USA) mAb and sorted with a FACS to obtain highly purified CD11c+ Gr-1− iDCs.

Flow cytometry

The mAb used for the experiments were as follows: anti-CD11c (PE-conjugated; eBioscience Inc.), anti-Gr-1 (FITC-conjugated; BD Biosciences), anti-CD86 (FITC-conjugated; BD Biosciences), anti-CD14 (PE-conjugated; eBioscience Inc.), anti-TLR4-MD2 (PE-conjugated; eBioscience Inc.), anti-RANKL/TNF-related activation-induced cytokine (PE-conjugated; eBioscience Inc.), anti-TNF-α (PE-conjugated; eBioscience Inc.), and anti-IL-12p40 (PE-conjugated; eBioscience Inc.). FITC and PE isotype controls were run in each experiment to determine the gating parameters. FACS data were analyzed using CellQuest software (BD PharMingen, San Diego, CA, USA).

Quantitative real-time PCR

Primers for RANKL were as follows: forward 5′-GGCCACAGCGCTTCTCAG-3′, reverse 5′-GAGTGACTTTATGGGAACCCGAT-3′. RNA was purified using the RNeasy Mini kit (Qiagen, Valencia, CA, USA). Relative quantitation of genes was performed using an Applied Biosystems 7300 machine. RANKL expression was determined using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s suggested protocol. The GAPDH gene was used as an endogenous control to normalize for differences in the amount of total RNA present in samples.

Cell death assay

Dead cells were detected by trypan blue exclusion and/or FACS analysis using cells incubated for 45 min at 37°C with 50 nM Mitotracker Red CMXRos (Invitrogen, Carlsbad, CA, USA). FACS data were analyzed using CellQuest software (BD Biosciences).

Cytokine assays

iDCs were incubated with 1 μg/ml E. coli LPS (Sigma-Aldrich, St. Louis, MO, USA) for 1, 3, 6, 12, or 24 h. In some experiments, graded doses of rOPG (∼1 μg/ml, RDI) and anti-OPG sera (∼10 μg/ml, R&D Systems, Minneapolis, MN, USA) were added to OPG KO and WT DC cultures, respectively, prior to the LPS stimulation for 24 h. Normal goat IgG (R&D Systems) was used as an isotype control. For investigation of in vivo cytokine production, mice received an i.p. administration of LPS (20 μg), and blood samples were collected 1 and 3 h after the LPS injection. The amounts of cytokines in cell culture supernatants and sera were determined by ELISA. The amounts of IL-6, IL-12p40, IL-12p70, TNF-α (R&D Systems), and IL-23 (eBioscience Inc.) were quantified with ELISA kits. For experiments involving intracellular staining of TNF-α and IL-12p40, cells were treated with 3 μg/ml brefeldin A (eBioscience Inc.) after LPS stimulation. Cells were permeablized following BD GolgiPlug (BD Biosciences) protocol, and the samples were analyzed using FACS and CellQuest software (BD Biosciences).

RESULTS

WT and OPG KO DCs express similar levels of RANKL and RANK

As RANKL and RANK have been reported to be coexpressed on a subset of human DCs [22], we examined first if mouse DCs could express RANKL. WT and OPG KO iDC expressed relatively little RANKL (Fig. 1A). LPS stimulation induced an increase in RANKL protein levels on WT and OPG KO DCs and also increased RANKL mRNA levels (e.g., Fig. 1B). In contrast, RANK was expressed on WT and OPG KO iDCs and did not change significantly after LPS stimulation (Fig. 1C).

Figure 1.

Figure 1.

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WT and OPG KO DCs express similar levels of RANKL and RANK. Expression of cell surface RANKL (A) and RANK (C) on WT and OPG KO DCs. Open histograms represent before and 24 h after LPS stimulation (1.0 μg/ml) determined using flow cytometry. Isotype controls are indicated with closed histograms. LPS induced increases in RANKL protein (A) as well as in RANKL mRNA in WT DCs as quantified by quantitative PCR (B) but did not alter RANK levels significantly (C).

OPG and Bim KO DCs survive longer than WT DCs

Next, we compared the spontaneous cell death of OPG KO DCs with WT DCs. After 24 h in culture, the viability of OPG KO DCs was consistently higher than WT DCs (Fig. 2), indicating that OPG KO DCs have a longer lifespan than WT DCs. We also examined whether OPG KO DCs resembled DCs missing the proapoptotic BH3-only protein, Bim. Like OPG KO DCs, Bim KO DCs were consistently more viable after culture than WT DCs (Fig. 2), as reported by others [13].

Figure 2.

Figure 2.

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Spontaneous cell death of iDCs obtained from WT, OPG KO, and Bim KO was determined by FACS using Mitotracker Red CMXRos. Representative results from at least three independent experiments are shown. *, P < 0.05.

Increased cytokine production by OPG KO DCs

To investigate the role of OPG in DC responses to pathogenic stimulus, we stimulated DCs from OPG KO or WT mice with E. coli LPS and measured cytokine production. Compared with WT DCs, OPG KO DCs secreted more TNF-α, IL-12p40, and IL-23 (Fig. 3A); however, LPS-stimulated OPG KO and WT DCs produced similar amounts of IL-6 and IL-12p70 (Fig. 3A). TNF-α and IL-23 were detectable in culture supernatants 1–3 h after LPS stimulation and reached maximum levels at 12 h. IL-12p40 and IL-6 were detectable somewhat later: IL-12p40 levels increased linearly for 24 h, and IL-6 levels reached a plateau at 12 h.

Figure 3.

Figure 3.

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OPG KO DCs after LPS stimulation produce more inflammatory cytokines than WT DCs. Secretion of TNF-α, IL-6, IL-12p70, IL-12p40, and IL-23p19 (A) by WT and OPG KO DCs treated with E. coli LPS (1 μg/ml) was measured by ELISA using supernatants harvested at indicated time-points. All assays were performed in triplicate to obtain mean ± sd. Secretion of TNF-α, IL-12p70, and IL-12p40 (B) and IL-23 (C) by WT and Bim KO DCs treated with E. coli LPS (1 μg/ml). Supernatants were harvested 24 h after the stimulation. (D) Levels of intracellular TNF-α and IL-12p40 in WT (closed histograms) and OPG KO (open histograms) DCs were compared using flow cytometry. DCs were treated with E. coli LPS (1 μg/ml) for 8 and 15 h followed by treatment with brefeldin A. Representative results from at least three independent experiments are shown in A and B. Means from three independent experiments are shown in C. *, P < 0.05; **, P < 0.01; N.S., not significant.

Like OPG KO DCs, Bim KO DCs produced more TNF-α and IL-12p40 than WT DCs but not more IL-12p70 (Fig. 3B). However, unlike OPG KO DCs, Bim KO DCs did not make more IL-23 than WT DCs (Fig. 3C). As OPG and Bim KO DCs survive longer, their selective increase in TNF-α and IL-12p40 may well be a result of their increased survival. However, the increase in IL-23 production by OPG KO DCs compared with WT and Bim KO DCs (Fig. 3C) is probably not simply a result of an increase in DC survival.

We also examined intracellular TNF-α and IL-12p40 levels in stimulated WT and OPG KO DCs to determine if per-cell production of cytokines was different. Although there was no difference in TNF-α production between WT and OPG KO DCs, OPG KO DCs secreted somewhat more IL-12p40 than WT DCs (Fig. 3D).

WT and OPG DCs express similar levels of CD14 and TLR4-MD2 complexes

Next, we assessed the potential mechanisms that might contribute to the enhanced cytokine production by OPG KO DCs. As DCs sense LPS with TLR4 complexes, we examined whether there were differences between OPG KO and WT DCs in their expression of CD14 or TLR4-MD2 complexes. Expression of CD14 (Fig. 4A) and TLR4-MD2 complexes (Fig. 4B) was similar on WT and OPG KO DCs before and after LPS stimulation.

Figure 4.

Figure 4.

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WT and OPG KO DCs express similar levels of CD14 and TLR4-MD2. Expression of CD14 (A) and TLR4-MD2 complexes (B; open histograms) in WT and OPG KO DCs before and after 24 h of LPS stimulation (1.0 μg/ml). Isotype controls (closed histograms) are also shown.

OPG regulates DC survival and cytokine production

To test if the increased TNF-α and IL-12p40 production by OPG KO DCs was a result of OPG, we added rOPG to OPG KO iDCs prior to LPS stimulation. Addition of graded doses of rOPG to OPG KO DC cultures decreased TNF-α and IL-12p40 production significantly after LPS stimulation (Fig. 5A, top). The addition of OPG also reduced DC survival significantly in the LPS-stimulated cultures (Fig. 5A, bottom). IL-23 production by highly purified, sorted OPG KO DCs also was decreased significantly in response to a graded dose of rOPG (Fig. 5B). In a converse experiment, we tested the effect of anti-OPG sera on cytokine production by and survival of WT DCs (Fig. 5C). The addition of anti-OPG sera to WT DC cultures enhanced IL-12p40 production and DC survival (Fig. 5C). These results show that OPG can regulate DC survival and cytokine production.

Figure 5.

Figure 5.

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OPG regulates DC survival and cytokine production. Secretion of TNF-α and IL-12p40 by OPG KO DCs and viability of OPG KO DCs treated with rOPG and E. coli LPS (1 μg/ml; A); secretion of IL-23 by OPG KO highly purified (sorted) DCs treated with rOPG and E. coli LPS (1 μg/ml; B); secretion of IL-12p40 by WT DCs and viability of WT DCs treated with anti-OPG serum or isotype control serum with E. coli LPS (1 μg/ml; C). DCs were cultured for 24 h, and supernatants were processed for ELISA. Viable cell numbers were determined by trypan blue dye exclusion. All assays were performed in triplicate to obtain mean ± sd. Representative results from at least three independent experiments are shown. *, P < 0.05; **, P < 0.01.

Elevated production of TNF-α by OPG KO mice following administration of LPS

To determine whether OPG KO mice have dysregulated cytokine production in vivo, we inoculated OPG KO and WT mice i.p. with a low dose of LPS; 1 or 3 h later, we bled the mice and measured serum levels of TNF-α IL-12p40, and IL-6. TNF-α was undetectable/below detection limits in sera from uninjected control animals (data not shown). More TNF-α was detected in OPG KO mouse serum samples taken 1 h after the LPS administration than in WT serum samples (Fig. 6); within 3 h after the LPS administration, TNF-α levels were reduced in WT and OPG KO mice. Similarly, after LPS inoculation, levels of IL-12p40 were higher in OPG KO mice (Fig. 6, middle). However, as seen in vitro with WT and OPG KO DCs (Fig. 3A), levels of IL-6 were not significantly different between WT and OPG KO mice (Fig. 6, bottom). Thus, LPS-induced cytokine production is selectively dysregulated in OPG KO mice.

Figure 6.

Figure 6.

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Induction of TNF-α, IL-12p40, and IL-6 in WT (open bars) and OPG KO (solid bars) mice, which were inoculated i.p. with LPS (20 μg), and blood samples were obtained at the indicated time-points. Serum TNF-α, IL-12p40, and IL-6 levels were measured by ELISA. All assays were performed in triplicate to obtain mean ± sd. Representative results from at least three independent experiments are shown. *, P < 0.05; **, P < 0.01.

DISCUSSION

We found that OPG regulates not only DC survival but also cytokine production by DCs. OPG KO DCs have better survival and produce more proinflammatory cytokines, including TNF-α, IL-12p40, and IL-23 in response to E. coli LPS than WT DCs. In addition, OPG KO DCs produce somewhat more IL-12p40 than WT DCs on a per-cell basis. This is most likely a result of the fact that the mouse DCs express RANKL and RANK so that RANKL-RANK interaction between DCs can occur. It is not simply a result of the differences in the expression of TLR4 complexes including CD14 between OPG KO and WT DCs. In support of a model for direct regulation of DCs by OPG, we found that adding back OPG to OPG KO DC cultures reduced DC survival and cytokine production to levels seen with WT DCs; furthermore, when we blocked OPG in WT DC cultures, DC survival and cytokine expression increased to levels seen with OPG KO DCs, which produce OPG after stimulation by, e.g., LPS or CD40 ligation [25, 26]. Thus, OPG produced by DCs or other cells may normally limit DC survival and TLR4-mediated cytokine production. This model is consistent with previous studies showing that RANKL can induce DC survival and cytokine secretion by DCs [19] and that DCs interact with each other in vivo [28]. It is also noteworthy that secretion of IL-6 was not affected by the absence of OPG in vitro and in vivo, indicating that OPG regulation of cytokine secretion in response to LPS may be selective. Our in vivo cytokine data are different than those in another study [29]. This difference might be a result of differences in the origin of LPS used (E. coli vs. Salmonella minnesota [29]) and/or the dose of LPS-given mice (20 μg in our study vs. 2 μg/g [29]).

Cytokines are critical mediators in autoimmune processes leading to, e.g., EAE [30, 31], RA [32], and psoriasis [33]. The dysregulation of cytokine production in OPG KO DCs may contribute directly to the diseases reported in OPG KO mice, such as cardiovascular diseases [24, 34]. The absence of TLR4 or MyD88 in atherosclerosis-prone ApoE KO mice leads to a reduction of the atherosclerotic plaque associated with reduced circulating levels of IL-12p40 [35]. Conversely, the high levels of proinflammatory cytokines seen in OPG KO mice in vitro and in vivo might be one of the key factors for predisposing OPG/ApoE KO mice further in the development of cardiovascular disease [33]. It will be interesting to determine if OPG KO mice are more susceptible to IL-23-associated diseases, such as EAE [30, 31], and infection by the bacterium Citrobacter rodentium [35], as OPG KO DCs produce more IL-23. OPG is also known to have therapeutic effects. Ashcroft et al. [36] reported that rOPG reverses skeletal abnormalities and reduces colitis by decreasing colonic DC numbers seen in IL-2 KO mice. Collectively, their data and our findings suggest that OPG prevents excessive inflammatory responses by limiting the DC lifespan.

It is well-established that OPG can inhibit osteoclastogenesis through direct blockade of RANKL-RANK interactions [37]. However, OPG may function indirectly to regulate osseous homeostasis through the inhibition of cytokine production. OPG may regulate the levels of proinflammatory cytokines in vivo and the amount and quality of cytokines DCs produce, such as TNF-α, which induces osteoclastogenesis independent of RANKL-RANK interaction [38]. OPG expression is markedly dropped in patients with RA [39], and TNF-α is produced mainly within the inflamed synovial tissue in RA [40]. Thus, OPG potentially may function not only to prevent RANKL-mediated osteoclastogenesis but also to inhibit bone-resorptive TNF-α production by DCs.

Bim KO DCs, like OPG KO DCs, produced more TNF-α and IL-12p40 and survive longer in culture. These results suggest that enhanced cytokine production could be a result of not only a better survival of Bim KO DCs [13] but also a regulation of certain cytokine production by a Bcl-2 family survival pathway in DCs This pathway is regulated by RANKL-RANK [9, 11, 20] and is dysregulated in the absence of Bim. Previous investigators reported [41] that DCs produce IL-12 transiently in response to LPS and then become refractory to further stimulation. This suggests that DCs with a longer lifespan may produce proinflammatory cytokines, such as IL-12 and TNF-α.

NF-κB p50/cRel double-KO DCs show impaired IL-12p40 expression in response to LPS, and p50 KO or cRel KO DCs show normal expression [21]. A recent study showed that LPS-stimulated cRel KO DCs are greatly impaired in their expression of IL-23p19 mRNA, indicating that cRel plays an essential role in IL-23p19 gene expression in DCs [42]. Further experiments are in progress to define more fully how RANK signaling in DCs regulates IL-23p19 expression. Therefore, taken together, RANK-RANKL interactions may selectively regulate production of not only IL-12/23p40 but also IL-23p19, and this may account for why OPG KO DCs produce more IL-23 than WT DCs.

In conclusion, OPG KO DCs survive better and produce more cytokine than WT DCs in response to LPS treatment. OPG functions not only as an “osteoprotective” but also as a regulatory of TLR-induced DC-associated inflammation. RANKL is up-regulated on DCs by LPS treatment, resulting in the RANKL-RANK interaction between DCs that synergistically functions with TLR signaling mediated by LPS. This RANKL-RANK interaction is limited by OPG. However, in the absence of OPG, RANK-RANKL interactions between DCs are sustained, leading to enhanced DC survival, cytokine production, and potentially, dysregulation of inflammatory responses.

ACKNOWLEDGMENTS

This study was supported by National Institutes of Health grants AI44257 and DE16381. We thank Mr. Shinji Kasahara for helping with DC sorting. We also thank Drs. Grant Hughes and Daniela Giordano for helpful discussions.

DISCLOSURE

The authors have no financial conflict of interest.

Footnotes

Abbreviations: ApoE=apolipoprotein E, CLR=C-type lectin receptor, DC=dendritic cell, EAE=experimental authoimmune encephalomyelitis, iDC=immature DC, KO=knockout, MD2=myeloid differentiation protein 2, OPG=osteoprotegerin, PAMP=pathogen-associated molecular pattern, RA=rheumatoid arthritis, RANKL=receptor activator of NF-κB ligand, WT=wild-type

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