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The Journal of Immunology, 2007, 179: 266-274.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Receptor Activator of NF-{kappa}B Ligand Inhibition Suppresses Bone Resorption and Hypercalcemia but Does Not Affect Host Immune Responses to Influenza Infection1

Robert E. Miller*, Daniel Branstetter{dagger}, Allison Armstrong*, Bryan Kennedy*, Jon Jones*, Laine Cowan{ddagger}, Jeanine Bussiere§ and William C. Dougall2,*

* Department of Cancer Biology, {dagger} Department of Pathology, and {ddagger} Toxicology, Amgen Washington, Seattle, WA 98119; and § Department of Toxicology, Amgen, Thousand Oaks, CA 91320


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Receptor activator of NF-{kappa}B (RANK) and its ligand (RANKL) are essential for osteoclast formation, function, and survival. Osteoprotegerin (OPG) inhibits RANK signaling by sequestering RANKL. This study evaluated the antiosteoclast and immunoregulatory effects of mouse rRANK-Fc, which, similar to OPG, can bind RANKL. The effect of RANKL inhibition by RANK-Fc on osteoclast function was determined by inhibition of vitamin D3 (1,25(OH)2D3)-induced hypercalcemia. Mice were injected with a single dose of 0, 10, 100, 500, or 1000 µg of RANK-Fc; 100 µg of OPG-Fc; or 5 µg of zoledronate 2 h before 1,25(OH)2D3 challenge on day 0, and sacrificed on days 1, 2, 4, 6, 8, 12, 16, and 20. RANK-Fc doses of 100 or 500 µg were tested in a mouse respiratory influenza virus host-resistance model. A single dose of RANK-Fc ≥100 µg suppressed elevation of serum calcium levels and suppressed the bone turnover marker serum pyridinoline at day 4 and later time points, similar to those observed with OPG-Fc and zoledronate (p ≤ 0.01 vs controls). By day 6, both immature and mature osteoclasts were depleted by high doses of RANK-Fc (500 and 1000 µg) or 100 µg of OPG-Fc. RANK-Fc doses of 100 or 500 µg had no detectable effect on immune responses to influenza infection, as measured by activation of cytotoxic T cell activity, influenza-specific IgG response, and virus clearance. RANK-Fc inhibition of RANKL has antiosteoclast activity at doses that have no detectable immunoregulatory activity, suggesting that RANKL inhibitors be further studied for their potential to treat excess bone loss.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bone remodeling is a dynamic process necessary for regulating bone structure and function that involves the coordinated function of osteoblasts and osteoclasts (1). Net resorption of bone occurs when there is an imbalance between synthesis and resorption, as is commonly found in various malignancies and postmenopausal osteoporosis (1). Osteoblasts are derived from mesenchymal precursors and promote bone synthesis by secreting a complex mixture of bone matrix proteins, known as osteoid, whereas osteoclasts are multinucleated cells derived from hemopoietic precursors and resorb mineralized bone matrix (2, 3).

The regulation of osteoclast differentiation is mediated by the receptor activator of NF-{kappa}B (RANK)3 ligand (RANKL), a member of the TNF superfamily of ligands, and two receptors, osteoprotegerin (OPG) and RANK (4). RANKL is expressed by osteoblasts and their precursors on the cell surface and induced during bone resorption. RANK is located on osteoclast progenitors. RANKL (also known as TNF-related activation-induced cytokine and osteoclast-differentiating factor) promotes osteoclast differentiation and activation and suppresses osteoclast apoptosis when bound to its cognate receptor RANK (4, 5, 6). RANK- and RANKL-deficient mice exhibit severe osteopetrosis, characterized radiographically by opacity in long bones, vertebral bodies, and ribs and by significantly increased total and trabecular bone density (7, 8).

Osteoblasts also secrete OPG, a soluble decoy receptor that binds to and sequesters RANKL, thereby preventing the activation of osteoclast differentiation and bone resorption. Overexpression of OPG or administration of rOPG has led to increases in bone density and protected against development of osteoporosis (9). In contrast, OPG-deficient mice developed early onset osteoporosis characterized by reductions in trabecular and cortical bone density and increased fracture incidence (10). Inhibition of RANKL by a rOPG fused to IgG1 Fc (OPG-Fc) has dramatically inhibited osteoclastogenesis and bone resorption in multiple models of bone disorders. In mice, a single dose of OPG-Fc inhibited osteoclasts for >6 days (11). Furthermore, OPG-Fc was effective in reversing humoral hypercalcemia of malignancies (HHM) in mouse models (12). Bisphosphonates are a standard treatment option for preventing bone loss in various settings, including HHM (13). OPG-Fc was superior to the bisphosphonate zoledronate in preventing hypercalcemia in these mouse models (12).

RANK-Fc has been shown to sequester RANKL in a manner similar to that of OPG, thereby inhibiting osteoclast formation, function, and survival. RANK-Fc suppressed bone resorption and prevented tumor-induced hypercalcemia (6, 14, 15, 16, 17).

The genetic ablation of RANKL and RANK activities in knockout mice revealed that these molecules may also influence embryonic development of the murine immune system. Knockout mice completely deficient in either RANKL or RANK lacked lymph nodes, yet developed normal splenic structure, Peyer’s patches, and dendritic cell function, and had normal survival (7, 8). RANKL knockout mice also showed defects in the early differentiation of T and B cells, whereas RANK knockout animals showed impaired B cell development, but normal T cell differentiation and activation (7, 8). OPG knockout mice showed increased differentiation of B cell progenitors and enhanced T cell stimulatory capacity of dendritic cells (18). Although administration of rOPG modestly stimulated the production of Ag-specific Abs against T cell-dependent and -independent Ags, it had no detectable effect on cellular immune responses. OPG had no detectable effect on cell-mediated reactions, including contact hypersensitivity, granuloma formation, and clearance of mycobacterial infection (19).

Immunoregulatory testing allows the evaluation of the effects of a compound on the immunocompetence of an animal to respond to a well-defined stimulus, such as infection or immunization (20), when the stimulus is provided postnatally. In the influenza virus host-resistance model, immunoregulatory effects are manifested by increased susceptibility toward infection (21, 22). The immune response to influenza virus involves production of immune mediators, enhancement of macrophage and NK cell activity, activation of CTL, and formation of Abs, and is manifested as changes in viral clearance, body weight, lung and spleen weight, cytokine production in the lung, and presence of Ag-specific IgG in the lung following inoculation (23).

The current studies were designed to compare the effects of RANKL inhibition by RANK-Fc with those of OPG-Fc and the bisphosphonate zoledronate in hypercalcemia, in addition to evaluating potential immunoregulatory effects of RANK-Fc in modulating influenza virus host resistance in a murine model. The data obtained from these studies indicate that RANKL inhibition causes a greater suppression of osteoclast activity compared with bisphosphonates and, moreover, selective blockade of RANKL (using RANK-Fc) had no detectable effect on immune responses in vivo.


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

C57BL/6 mice were obtained from Taconic Farms. Female mice between 10 and 12 wk of age were used for the vitamin D3 (1,25(OH)2D3) model, and male and female mice at 6 wk of age were acclimated for 18 days before use in the influenza virus host-resistance model. Purina Rodent Chow 5002 (Ralston Purina) or Harlan Teklad Rodent Diet 8728C and tap water were provided ad libitum. The animals were observed daily to twice daily for mortality and moribundity. All experiments performed at Amgen were approved and performed in accordance with guidelines set out by the Amgen Animal Use and Care Committee.

Test compounds

RANK-Fc and OPG-Fc were stored frozen in vials at –60°C to –80°C until use. rRANK-Fc is a fusion protein containing aa 1–213 of the murine RANK extracellular domain with the C terminus of the Fc domain of murine IgG1. The recombinant protein was produced from Chinese hamster ovary cells. On the day of dosing, the vials were thawed and the contents were diluted in PBS. RANK-Fc was prepared in concentrations of 0.05, 0.5, 2.5, and 5 mg/ml for the 1,25(OH)2D3 hypercalcemia model and concentrations of 0.5 and 2.5 mg/ml for use in the influenza virus host-resistance model. A dose volume of 0.2 ml was administered s.c.; OPG-Fc (24) concentration was 0.5 mg/ml. Each vial was thawed only once.

The 1,25-(OH)2D3 (Sigma-Aldrich) was dissolved in 5% alcohol and 95% corn oil (Sigma-Aldrich). Zoledronate (Novartis Pharmaceuticals) was suspended in PBS and injected once into the tail vein at a dose of 5 µg. Dexamethasone-21 phosphate was dissolved at a concentration of 0.5 mg/ml in a vehicle consisting of 0.5% methylcellulose/0.2% Tween 80 in distilled water, then administered daily by oral gavage at a dose of 5 mg/kg in a volume of 10 ml/kg. Mice were weighed twice weekly to ensure delivery of the indicated dose.

On a molar basis, the dose of RANK-Fc or OPG-Fc is equivalent to zolendronate. Zolendronate has a molecular mass of 290.1 g/mol, and therefore, the 5 µg used per mouse is equal to 17.2354 nmol/mouse. RANK-Fc (as a monomer) has a molecular mass of 55 kDa; therefore, at the highest dose used (1000 micrograms/mouse), this amount is equal to 18.1818 nmol/mouse.

The 1,25(OH)2D3 hypercalcemia model

A total of 192 C57BL/6 mice was randomly assigned to six groups (24 mice in each dose group and control group). Acute hypercalcemia was induced by challenge, injecting 0.5 µg of 1,25(OH)2D3 s.c. in a volume of 50 µl on days 0–4. Each mouse received a single dose of 0, 10, 100, 500, or 1000 µg of RANK-Fc; 100 µg of OPG-Fc; or 5 µg of zoledronate 2 h before challenge on day 0 with or without 1,25(OH)2D3. These mice were fed a low-calcium diet (0.02% calcium; PMI Feeds) from 3 days before dosing until 3 days after 1,25(OH)2D3 challenge.

Mice treated with 1,25(OH)2D3, but without other test articles, became moribund after day 5 and were sacrificed in accordance with Institutional Animal Care and Use Committee guidelines. Groups of three mice, each receiving single doses of RANK-Fc, OPG-Fc, or zoledronate, remained in good physical condition and were sacrificed on days 1, 2, 4, 6, 8, 12, 16, and 20, after which serum markers and skeletal parameters were analyzed. Blood was collected before the first dose of 1,25(OH)2D3 and 3 h after the dosing. Blood-ionized calcium levels were determined by Ani Lytics. Pyridinoline (PYD) peptide cross-links of type I collagen were collected as a specific biomarker for bone turnover. Serum PYD levels were determined (Metra Serum PYD ELISA catalogue 8019; Quidel). Animals were necropsied after asphyxiation with CO2. The necropsy included a macroscopic examination of the external features of the carcass, all external orifices, and abdominal and thoracic cavities, organs, and tissues. Body, lung, and spleen weights were recorded, and samples were stored at –20°C. At necropsy, femurs, tibias, and lumbar vertebrae were collected and fixed in neutral buffered formalin for histologic analysis.

Formalin-fixed tibias and lumbar vertebrae were decalcified and embedded in paraffin. Longitudinal sections were obtained from the proximal tibia metaphysis and from the vertebral body sagittal center and stained with H&E, and for tartrate-resistant acid phosphatase (TRAP) activity (leukocyte acid phosphatase kit; Sigma-Aldrich). Histomorphometric evaluations were performed in a blinded manner. The total area analyzed was 1.10 mm2 for each section. Cancellous bone criteria were evaluated, as previously described (25), using OsteoMeasure software (OsteoMetrics). Static histomorphometric parameters were calculated according to the recommendations of the American Society for Bone and Mineral Research committee (26).

Influenza virus host-resistance model

To test the effect of RANKL inhibition on the adult immune response to viral infection, multiple host-resistance parameters in response to mouse influenza virus challenge were evaluated after treatment with two doses of RANK-Fc.

A total of 260 mice was randomly assigned to six groups. A naive control group, consisting of 10 untreated and uninfected mice (5 males, 5 females), was necropsied on day –1 for baseline values. For comparison, another group of 10 uninfected mice received a single 500 µg dose of RANK-Fc on day –1 and was necropsied 4 h later. The remaining groups were anesthetized with isoflurane on day 0, and then infected intranasally with mouse-adapted influenza (~4 x 103 PFU in 50 µl Eagle’s MEM (E-MEM)). Three groups received treatment with 100 µg of RANK-Fc, 500 µg of RANK-Fc, or PBS vehicle control given s.c. on days –3 and –1 before virus infection and on days 2, 6, 9, 13, 16, and 20 following virus infection. The final group received 5 mg/kg dexamethasone by oral gavage starting on day –3 and continuing daily (except day 0) through day 20. Ten infected mice (5 males, 5 females) from each treatment group were sacrificed on days 2, 6, 8, 10, 14, and 21 for necropsy. Mice scheduled for necropsy on days 2 and 6 received RANK-Fc ~4 h before sacrifice, whereas dexamethasone was not administered on the day of sacrifice. Animals were necropsied, as described above. Body, lung, and spleen weights were recorded. In the influenza virus host-defense model, the lungs were homogenized immediately in E-MEM (5% w/v) and centrifuged, and samples were aliquoted into labeled cryotubes. Samples for testing of infectious virus were stored below –70°C. All other samples were stored at –20°C.

Infectious virus titer was determined in Madin-Darby canine kidney cells. Briefly, confluent cultures were incubated with 0.1 ml of lung homogenate or stock virus for 30–60 min and then covered with agarose. The monolayers were fixed with 10% buffered formalin and stained with crystal violet, and plaques were counted visually using a plaque viewer. Influenza-specific IgG levels in serum samples were determined using established immunochemical reactions in microplates coated with 0.5 µg/well influenza A/Port Chalmers/1/72 (H3N2) in PBS and measured using a SpectraMax 340 microplate reader (Molecular Devices). The assay that was used measures specific IgG Ab to influenza A/Port Chalmers/1/73 (H3N2). The concentrations of murine IL-1beta, IL-6, and TNF-{alpha} in the lung homogenates were determined using commercial murine ELISA kits (Pierce and R&D Systems). Right knee joints were collected from all mice on the day-21 necropsy, fixed in 10% buffered formalin, decalcified in 10% EDTA, and embedded in paraffin. The sections were TRAP stained by standard methods and evaluated for the presence of osteoclasts by using a 0–5 scoring system. A score of 0 indicated the absence of osteoclasts, and a score of 5 indicated the presence of numerous osteoclasts with extensive vacuolated cytoplasm located in resorption pits along bony trabeculae and the remodeling face of the growth plate. The thickness of the growth plate was measured using an ocular micrometer.

To confirm that treatment with RANK-Fc inhibited osteoclastogenesis over the prolonged time course necessary to monitor the host response to influenza virus, longitudinal changes in serum levels of the osteoclast marker TRAP5b (a bone turnover marker) and bone mineral density (BMD) were measured in an additional cohort of mice (n = 6/group) treated with the same dose and schedule of RANK-Fc as the virus-challenged mice.

Sera were harvested on days 9 and 26 after treatment initiation (n = 3/group/time point). Terminal blood samples were collected by cardiac puncture and at additional time points by retro-orbital bleeds. Samples from multiple time points were assayed for serum TRAP5b levels by ELISA using MouseTRAP assay (distributed by Immunodiagnostic Systems). Changes in BMD were measured by dual-energy x-ray absorptiometry (GE Lunar Piximus II; GE Healthcare).

Statistics

Data from the 1,25(OH)2D3 hypercalcemia model were assessed for the statistical significance of differences between groups for different time points using GraphPad Prism version 4.01 (GraphPad). Comparisons were made using Student’s t test for comparison of data from two groups, Dunnett’s test for comparison of multiple treatment groups with a control, and the Tukey-Kramer test for comparisons between multiple treatment groups or controls. The latter two methods make allowance for multiple comparisons. A p value of <0.05 was considered indicative of a statistically significant difference. Data from the influenza virus host-resistance model were analyzed using a general linear model approach for each gender separately and for both genders combined. A parametric multiple-regression model was used to test for significant fit for each parameter, with terms for treatment, gender, days, and one-way and two-way interactions. If a significant model fit was obtained, a two-sided Dunnett’s test of treatment was performed. Pairwise testing of treatment vs control was adjusted for multiple testing to achieve an experiment-wide {alpha} level of 0.05. If a significant model fit was not obtained, then it was assumed that there was no difference between treatments for these parameters. Significance was achieved at a two-tailed probability level of 0.05. Body weight, food consumption, and clinical pathology data were evaluated using descriptive statistics.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The 1,25(OH)2D3 model

Whole-blood ionized calcium levels were elevated above those of controls by day 4 following injection with 1,25(OH)2D3 (Fig. 1A) and continued to increase at days 6 and 8 (data not shown). Treatment with each dose of RANK-Fc in combination with 1,25(OH)2D3 significantly reduced the hypercalcemia at day 4 (Fig. 1A) and maintained serum calcium levels in the normal range by day 6 through day 20 (data not shown). Single doses of RANK-Fc 500 µg or higher suppressed the 1,25(OH)2D3-induced elevation of serum calcium at day 4 and later time points, compared with 1,25(OH)2D3-only controls (p < 0.001).


Figure 1
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  		FIGURE 1. Alterations in serum measurements of bone turnover in 1,25(OH)2D3-challenged mice treated with RANK-Fc or OPG-Fc. A, Serum calcium responses in 1,25(OH)2D3-challenged mice treated with RANK-Fc or OPG-Fc. Mice receiving a low-calcium diet were treated with vehicle only (low-Ca2+ food), challenged with either 1,25(OH)2D3 only (1,25(OH)2D3 control) for 5 days or 1,25(OH)2D3 in combination with a single dose of 50, 100, 500, or 1000 µg of RANK-Fc; 100 µg of OPG-Fc; or 5 µg of zoledronate, given 2 h before 1,25(OH)2D3 challenge (n = 3/group). Mean serum calcium concentrations (mg/dl) are expressed with SDs. Treatment of mice on a low-calcium diet with 1,25(OH)2D3 induces hypercalcemia (>11 mg/dl), which is reduced by all RANK-Fc doses, OPG-Fc, and zoledronate (*, p < 0.001 compared with controls; Tukey’s multiple comparison test). B, Serum PYD responses in 1,25(OH)2D3-challenged mice treated with RANK-Fc or OPG-Fc. Mice receiving a low-calcium diet were treated with vehicle only (low-Ca2+ food), challenged with either 1,25(OH)2D3 only (,1,25(OH)2D3 control) for 5 days or 1,25(OH)2D3 in combination with a single dose of 50, 100, 500, or 1000 µg of RANK-Fc; 100 µg of OPG-Fc; or 5 µg of zoledronate given 2 h before 1,25(OH)2D3 challenge (n = 3/group). Mean serum PYD concentrations (nmM/l) are expressed with SDs. Treatment of mice on a low-calcium diet with 1,25(OH)2D3 induces a dramatic increase of serum PYD bone turnover marker, which is prevented by all RANK-Fc doses, OPG-Fc, and zoledronate (*, p < 0.01 compared with controls; Tukey’s multiple comparison test).

 
A single dose of zoledronate (5 µg) also suppressed the hypercalcemia, starting at day 4 and continuing until end of treatment. PYD, a more specific serum marker of bone turnover than calcium, was significantly elevated after 1,25(OH)2D3-induced bone resorption in control mice by day 4 of treatment (Fig. 1B). Consistent with the prevention of hypercalcemia, all dose levels of RANK-Fc, as well as OPG-Fc and zoledronate, suppressed 1,25(OH)2D3-dependent induction of this marker from day 4 until the end of treatment, compared with 1,25(OH)2D3 only (p < 0.01).

Histologic analysis of the distal femur and lumbar vertebrae demonstrated that 1,25(OH)2D3 treatment induced bone loss, characterized by an increased number of TRAP-positive osteoclasts and an increased degree of osteoclast contact with trabecular bone (Fig. 2A). Morphologic changes consistent with osteoclast apoptosis, including marked karyorexis or fragmentation and condensation of nuclei, were evident 24 h after treatment with 500 or 1000 µg of RANK-Fc or 100 µg of OPG-Fc. By day 2, these morphologic changes in osteoclasts were also evident in animals treated with the lower doses of RANK-Fc (50 or 100 µg) and zoledronate (data not shown). At day 4 of treatment, both immature and mature osteoclasts were completely depleted by high doses (500 or 1000 µg) of RANK-Fc or the single 100 µg dose of OPG-Fc (Figs. 2A and 3A). Inhibition of osteoclastogenesis was sustained beyond 12 days after a single dose of either 1000 µg of RANK-Fc or 100 µg of OPG-Fc (Figs. 2B and 3B), followed by a return to normal osteoclast values by day 20 (data not shown). In contrast, a single dose of zoledronate only eliminated a subset of osteoclasts (illustrated by histologic analysis in Fig. 2 and quantified in Fig. 3B). Significant numbers of TRAP-positive cells were evident following zoledronate treatment, which suggests resistance to treatment with zoledronate throughout the study period. The inhibition of osteoclast formation observed at day 4 after treatment with RANK-Fc or OPG-Fc was associated with increases in the femoral growth plate thickness (Fig. 2) and a significant decrease in the percentage of osteoclast surface area relative to total bone surface area (% OcS/BS) (p < 0.01; Fig. 3A). At day 12, the osteoclast surface of 1,25(OH)2D3-challenged mice was significantly reduced by 50 µg of RANK-Fc and doses greater than 100 µg and by OPG-Fc compared with 1,25(OH)2D3-only controls (p < 0.001; Fig. 3B).


Figure 2
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  		FIGURE 2. Representative photomicrographs of the proximal tibia metaphysis in hypercalcemic mice treated with RANK-Fc, OPG-Fc, or zoledronate. Mice receiving a low-calcium diet were treated with vehicle only (low-Ca2+ food), challenged with either 1,25(OH)2D3 only (1,25(OH)2D3 control) for 5 days or 1,25(OH)2D3 in combination with a single dose of 1000 µg of RANK-Fc; 100 µg of OPG-Fc; or 5 µg of zoledronate given 2 h before 1,25(OH)2D3 challenge. Sections were stained for TRAP (red) to highlight osteoclasts. A, Representative photomicrographs of TRAP-positive osteoclasts at day 4 after treatment; B, representative photomicrographs of TRAP-positive osteoclasts at day 12 after treatment. Treatment of mice on a low-calcium diet with 1,25(OH)2D3 induced a dramatic increase in the intensity of TRAP-staining osteoclasts at the growth plate. At days 4 and 12, 100 µg of OPG-Fc or 1000 µg of RANK-Fc completely depleted the growth plate of osteoclasts compared with zoledronate. Note that zoledronate did not completely eliminate osteoclasts from the metaphysis at either day 4 or day 12.

 

Figure 3
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  		FIGURE 3. Bone histomorphometry of the proximal tibial metaphysis in mice challenged with 1,25(OH)2D3 and treated with RANK-Fc, OPG-Fc, or zoledronate. Mice receiving a low-calcium diet were treated with vehicle only (low-Ca2+ food), challenged with either 1,25(OH)2D3 only (1,25(OH)2D3 control) for 5 days or 1,25(OH)2D3 in combination with a single dose of either 50, 100, 500, or 1000 µg of RANK-Fc; 100 µg of OPG-Fc; or 5 µg of zoledronate given 2 h before 1,25(OH)2D3 challenge (n = 5/group). At sacrifice, tibias were harvested for histomorphometry. Data were not available from mice challenged with 1,25(OH)2D3 for 12 days without additional treatment due to extensive mortality. Data are expressed as osteoclast surface as a percentage of total bone surface (OcS/BS) with SEM at day 4 after treatment (A), and day 12 after treatment (B) (*, p < 0.001; **, p < 0.01; one-way ANOVA with Tukey’s multiple comparison test).

 
The sustained efficacy of RANKL inhibition to reduce the percentage of osteoclast-covered bone surface contrasted with no reduction of this parameter by zoledronate at day 12 (Fig. 3B). Control animals with hypercalcemia induced by 1,25(OH)2D3 also had profound weight loss. Treatment with either RANK-Fc, OPG-Fc, or zoledronate protected against the weight loss induced by 1,25(OH)2D3. Each test compound (OPG-Fc and zoledronate) was also associated with normalized calcium levels (data not shown), suggesting that the weight loss observed was a result of the hypercalcemia.

Influenza virus host-resistance model

Virus clearance measured per lung or per gram of lung tissue did not differ between mice treated with RANK-Fc at doses of 100 or 500 µg and those receiving vehicle control, whereas dexamethasone caused reduced viral clearance (p < 0.05) when compared with vehicle (Fig. 4A). By day 8, the log virus titer was 0.2 PFU/lung in the 100 µg RANK-Fc group and 0 PFU/lung in the 500 µg RANK-Fc and vehicle-control groups. In comparison, treatment with dexamethasone prolonged virus infection compared with vehicle control in males and females analyzed together (p < 0.05). The log virus titer was 2.0 PFU/lung on day 8, then declined to 0.3, and finally to 0 PFU/lung on days 10 and 14, respectively. The production of influenza-specific IgG increased normally following viral infection in animals receiving RANK-Fc as compared with those receiving vehicle (Fig. 4B). In contrast, dexamethasone significantly delayed and blunted the rise in influenza-specific IgG (p < 0.05) when compared with vehicle. These profiles were seen for males and females separately and for males and females combined.


Figure 4
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  		FIGURE 4. Mean viral clearance and influenza-specific IgG production in mice infected with the influenza virus and treated with RANK-Fc or dexamethasone. Mice were infected with the influenza virus, and host resistance was assessed by injecting vehicle, RANK-Fc, or dexamethasone (n = 10/group). A, Viral clearance measured as mean log virus titer/lung was similar in vehicle- and RANK-Fc-treated mice, with a significantly delayed clearance in dexamethasone-treated mice compared with those given vehicle (p < 0.05; two-sided Dunnett’s test). B, Influenza-specific IgG was detected at similar levels in vehicle- and RANK-Fc-treated mice compared with a statistically significant reduction in the dexamethasone-treated mice compared with vehicle (p < 0.05; two-sided Dunnett’s test).

 
IL-1beta, TNF-{alpha}, and IL-6 levels in lung homogenates (5% w/v) did not differ between naive controls and uninfected mice that received a single RANK-Fc dose (Fig. 5). The concentration of each cytokine increased following viral infection, with peak levels measured on day 6. Thereafter, cytokine levels declined and approached those found in the naive controls. Treatment with RANK-Fc or dexamethasone did not affect the virus-induced increase in IL-1beta or TNF-{alpha} in males or females separately or in males and females analyzed together. IL-6 levels were elevated slightly in the groups treated with RANK-Fc compared with vehicle controls, but these effects were not statistically significant. However, treatment with dexamethasone significantly increased IL-6 production when compared with vehicle controls in females separately and males and females analyzed together (p < 0.05).


Figure 5
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  		FIGURE 5. Lung cytokine levels in the influenza host-resistance model. Each point represents mean treatment group weight in grams ± SEM. Mice were infected with the influenza virus, and host resistance was assessed by injecting vehicle, RANK-Fc, or dexamethasone (n = 10/group). At day 21, the lungs were homogenized immediately in E-MEM (5% w/v) and centrifuged, and the concentrations of murine IL-1beta, IL-6, and TNF-{alpha} in the lung homogenates were determined using commercial murine ELISA kits. RANK-Fc had no significant effect on levels IL-1beta, IL-6, and TNF-{alpha} in the lung, whereas dexamethasone-treated mice had an increase in IL-6 at days 6 and 10 (p < 0.05; two-sided Dunnett’s test).

 
Body weight changes were observed following influenza infection, but did not differ between mice treated with 100 or 500 µg of RANK-Fc and those in the vehicle-control group (Fig. 6A). No sex-related differences in the weight profiles were observed. In contrast, dexamethasone significantly reduced body weight as compared with vehicle control (p < 0.05). The effect of dexamethasone on weight loss after infection was greater in males (mean loss of 23.4% body weight) than in females (mean loss of 19.0% body weight).


Figure 6
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  		FIGURE 6. Mean changes in body, lung, and spleen weight in mice infected with the influenza virus and treated with RANK-Fc or dexamethasone. Each point represents mean treatment group weight in grams ± SEM. Mice were infected with the influenza virus, and host resistance was assessed by injecting vehicle, RANK-Fc, or dexamethasone (n = 10/group). Body (A), lung (B), and spleen (C) weights (g) are expressed as means. RANK-Fc had no effect on mean body, lung, or spleen weight. Dexamethasone-treated mice had a decreased body weight starting on day 6, decreased lung weight starting on day 10, and decreased spleen weight starting on day 2 (p < 0.05; two-sided Dunnett’s test).

 
Influenza infection produced an increase in lung and spleen weights (Fig. 6, B and C). The lung and spleen weights of mice treated with RANK-Fc did not differ from those of mice in the vehicle-control group. In contrast, dexamethasone significantly reduced lung weight and markedly reduced spleen weight relative to vehicle control (both p < 0.05). This profile was seen for males and females separately and for males and females analyzed together.

Significant reductions in serum TRAP5b levels were observed with both doses of RANK-Fc tested, and this effect lasted for >3 wk (Fig. 7A). The suppression of this systemic marker of bone turnover was consistent with the significant increases in BMD observed after treatment with both doses of RANK-Fc at days 9 and 26 (Fig. 7B). In addition, analysis of osteoclasts in influenza-bearing mice treated with RANK-Fc demonstrated decreases in TRAP5b-positive osteoclasts in the proximal tibia and marked increases in the thickness of the growth plate of the proximal tibia by day 21 (Fig. 7C). The increase in growth plate thickness reflects the prolonged decrease in osteoclast-mediated remodeling of nascent trabeculae at the degenerative face of the growth plate. Dexamethasone produced a small decrease in osteoclasts in male mice at day 21, but otherwise had a negligible effect in this assessment as compared with vehicle controls (data not shown).


Figure 7
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  		FIGURE 7. Longitudinal analyses of osteoclast inhibition with RANK-Fc. Mice (n = 3/group) were treated with the same dose and schedule as the virally challenged mice. A, Serum samples were collected at multiple time points and frozen at –80°C. TRAP5b levels were determined by ELISA. Statistically significant decreases in TRAP5b were observed at both doses of RANK-Fc compared with PBS control (*, p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test). B, BMD changes were assessed in mice by dual-energy x-ray absorptiometry. Statistically significant increases in BMD were observed after treatment with both doses of RANK-Fc at days 9 and 26 compared with PBS control (*, p < 0.05; one-way ANOVA with Bonferroni’s multiple comparison test). C, Representative photomicrographs of TRAP-positive osteoclasts at day 21 after treatment. Treatment of influenza-bearing mice with 100 or 500 µg of RANK-Fc reduced the numbers of TRAP-positive osteoclasts and resulted in a marked increase in the thickness of the growth plate.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
These studies indicate that acute RANKL inhibition via the recombinant fusion protein RANK-Fc has pronounced antiosteoclast activities, but has minimal effects on immunity in vivo. The treatment of C57BL/6 mice with 1,25(OH)2D3 led to significant elevations in serum calcium levels by day 4, similar to the results observed previously with BDF1 mice (24). In the current study, a single injection of RANK-Fc at a dose as low as 100 µg prevented hypercalcemia and inhibited increases of PYD and osteoclast-dependent bone resorption as well as bone loss-dependent decreases in body weight. Concentrations below 100 µg did not achieve complete inhibition of markers of bone loss in this model and were not used in subsequent experiments. RANK-Fc doses of 100, 500, and 1000 µg were similarly effective in all measures of bone loss inhibition, with the exception of a more complete depletion of osteoclasts at the growth plate being demonstrated for 500 and 1000 µg of RANK-Fc. RANK/RANKL-dependent bone resorption has been observed in conjunction with hypercalcemia caused by 1,25(OH)2D3 treatment or by tumor-associated parathyroid hormone-related hormone in HHM models, as evidenced by calcium levels, biochemical markers of bone loss (including PYD), weight loss, and bone radiography (12, 15, 24). Importantly, as could be expected from the mechanism of action of RANK-Fc and OPG-Fc, both compounds had similar inhibitory effects on bone loss in the current study model.

This study demonstrated a lack of detectable immunoregulatory effects of RANKL inhibition (using RANK-Fc) on a wide range of immune parameters at dose levels that effectively eliminated osteoclasts and inhibited bone loss. This was evidenced by unchanged coordinated responses of a wide variety of immune system cells and components in the influenza virus host-defense model. In the influenza virus host-resistance model, immunoregulatory effects are evidenced by impaired clearance or elimination of the virus.

Clearance of influenza and other infectious agents is mediated through a cascade of immunologic mechanisms that function in a coordinated, time-dependent manner and depend on the two arms of the immune system, innate immunity and adaptive immunity (27). Innate immunity is the nonspecific immune response that involves activation of macrophages and NK cells, and production of IFNs and a variety of other cytokines. Adaptive immunity is the specific immune response that involves activation of CTL and production of specific Abs. Although we did not monitor effects of RANK-Fc on IFN production or NK cell and macrophage function directly in the current study, we did demonstrate that RANK-Fc did not affect clearance of the influenza virus or formation of influenza-specific IgG. These results in the flu-virus challenge model are consistent with the lack of any obvious macrophage or NK cell deficiencies in the RANK or RANKL knockout mice (7, 8). In addition, RANK-Fc did not detectably affect body, lung, or spleen weight, and it produced no significant changes in lung cytokine levels.

The observations that knockout mice completely lacking RANKL or RANK show an absence of lymph nodes and impaired lymphocyte differentiation (7, 8) suggest that the absence of RANK-mediated signaling may be associated with the embryonic development of these systems. However, this study indicates that RANKL inhibition (using RANK-Fc) does not detectably affect innate or adaptive immune responses in this particular model in adult animals. Future studies would be useful to address any contributions of this pathway during lethal viral challenges. The sensitivity of this influenza model to immunoregulatory effects was evidenced by the use of dexamethasone as a control for identifying immunoregulatory effects. Corticosteroids have been demonstrated to inhibit T cell function and reduce B cell Ig production, and have been associated with increased infection risk in some clinical settings (28). In the current study, dexamethasone delayed viral clearance, blunted influenza-specific IgG production, and reduced lung and spleen weights when compared with the vehicle. Of note, in addition to inhibiting osteoclast-mediated bone loss in the 1,25(OH)2D3 hypercalcemia model, RANK-Fc inhibition of RANKL significantly reduced osteoclast activity and correspondingly increased growth plate thickness in proximal tibias in the influenza virus host-defense model at concentrations lacking detectable immunoregulatory effects within the time frame of the study.

An interesting aspect of this study was the difference between the effects of RANK-Fc and OPG-Fc and those of bisphosphonates on the inhibition of osteoclast differentiation in the current model. RANK-Fc, OPG-Fc, and zoledronate demonstrated similar levels of inhibition of hypercalcemia, the bone resorption marker PYD, and weight loss. However, zoledronate did not achieve the same level of osteoclast depletion at the growth plate and bone surfaces as did RANK-Fc and OPG-Fc. This was the first direct comparison of RANK-Fc with bisphosphonates that paralleled the effects of OPG-Fc seen in comparison with a bisphosphonate (12).

These data provide evidence of unique and potentially therapeutic properties of inhibiting the RANK/RANKL system. Specifically, RANKL is required for osteoclast differentiation, activation, and survival, whereas bisphosphonate treatment does not affect osteoclast differentiation. Not only is the dosage of parenteral bisphosphonates limited by renal toxicity (29), but bisphosphonate resistance has been observed in some patients, indicating that an unmet medical need exists for an alternative treatment for bone resorption pathologies (30, 31). The unique mechanism of action and therapeutic potential of RANK/RANKL inhibition may provide alternative and more potent therapies for bone loss across a wide variety of pathologies, including postmenopausal osteoporosis, inflammatory bone diseases, bone loss due to hormone ablation, and cancer-induced bone disease.

In conclusion, these studies indicate that RANKL inhibition with RANK Fc, administered at concentrations effective in modulating bone metabolism, lacks detectable immunoregulatory activity in certain model systems and is unlikely to compromise the immunologic host defense against viral infections.


   Acknowledgments
 
We acknowledge Christine Gatchalian and Holly Zoog for editorial assistance on this manuscript and Burleson Research Technologies for conduct of the mouse viral influenza model.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
All authors are employees of and shareholders in Amgen.


   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 Amgen. Back

2 Address correspondence and reprint requests to Dr. William C. Dougall, Amgen Washington, 1201 Amgen Court West, Seattle, WA 98119-3105. E-mail address: dougallw{at}amgen.com Back

3 Abbreviations used in this paper: RANK, receptor activator of NF-{kappa}B; BMD, bone mineral density; E-MEM, Eagle’s MEM; HHM, humoral hypercalcemia of malignancies; OPG, osteoprotegerin; PYD, pyridinoline; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase. Back

Received for publication March 8, 2006. Accepted for publication April 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Boyle, W. J., W. S. Simonet, D. L. Lacey. 2003. Osteoclast differentiation and activation. Nature 423: 337-342. [Medline]
  2. Mackie, E. J.. 2003. Osteoblasts: novel roles in orchestration of skeletal architecture. Int. J. Biochem. Cell Biol. 35: 1301-1305. [Medline]
  3. Suda, T., N. Takahashi, N. Udagawa, E. Jimi, M. T. Gillespie, T. J. Martin. 1999. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev. 20: 345-357. [Abstract/Free Full Text]
  4. Hofbauer, L. C., M. Schoppet. 2004. Clinical implications of the osteoprotegerin/RANKL/RANK system for bone and vascular diseases. J. Am. Med. Assoc. 292: 490-495. [Abstract/Free Full Text]
  5. Anderson, D. M., E. Maraskovsky, W. L. Billingsley, W. C. Dougall, M. E. Tometsko, E. R. Roux, M. C. Teepe, R. F. DuBose, D. Cosman, L. Galibert. 1997. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390: 175-179. [Medline]
  6. Hsu, H., D. L. Lacey, C. R. Dunstan, I. Solovyev, A. Colombero, E. Timms, H. L. Tan, G. Elliott, M. J. Kelley, I. Sarosi, et al 1999. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc. Natl. Acad. Sci. USA 96: 3540-3545. [Abstract/Free Full Text]
  7. Dougall, W. C., M. Glaccum, K. Charrier, K. Rohrbach, K. Brasel, T. De Smedt, E. Daro, J. Smith, M. E. Tometsko, C. R. Maliszewski, et al 1999. RANK is essential for osteoclast and lymph node development. Genes Dev. 13: 2412-2424. [Abstract/Free Full Text]
  8. Kong, Y. Y., H. Yoshida, I. Sarosi, H. L. Tan, E. Timms, C. Capparelli, S. Morony, A. J. Oliveira-dos-Santos, G. Van, A. Itie, et al 1999. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397: 315-323. [Medline]
  9. Simonet, W. S., D. L. Lacey, C. R. Dunstan, M. Kelley, M. S. Chang, R. Luthy, H. Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al 1997. Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89: 309-319. [Medline]
  10. Bucay, N., I. Sarosi, C. R. Dunstan, S. Morony, J. Tarpley, C. Capparelli, S. Scully, H. L. Tan, W. Xu, D. L. Lacey, et al 1998. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 12: 1260-1268. [Abstract/Free Full Text]
  11. Lacey, D. L., H. L. Tan, J. Lu, S. Kaufman, G. Van, W. Qiu, A. Rattan, S. Scully, F. Fletcher, T. Juan, et al 2000. Osteoprotegerin ligand modulates murine osteoclast survival in vitro and in vivo. Am. J. Pathol. 157: 435-448. [Abstract/Free Full Text]
  12. Morony, S., K. Warmington, S. Adamu, F. Asuncion, Z. Geng, M. Grisanti, H. L. Tan, C. Capparelli, C. Starnes, B. Weimann, et al 2005. The inhibition of RANKL causes greater suppression of bone resorption and hypercalcemia compared with bisphosphonates in two models of humoral hypercalcemia of malignancy. Endocrinology 146: 3235-3243. [Abstract/Free Full Text]
  13. Body, J. J., R. Bartl, P. Burckhardt, P. D. Delmas, I. J. Diel, H. Fleisch, J. A. Kanis, R. A. Kyle, G. R. Mundy, A. H. Paterson, R. D. Rubens. 1998. Current use of bisphosphonates in oncology: International Bone and Cancer Study Group. J. Clin. Oncol. 16: 3890-3899. [Abstract/Free Full Text]
  14. Childs, L. M., E. P. Paschalis, L. Xing, W. C. Dougall, D. Anderson, A. L. Boskey, J. E. Puzas, R. N. Rosier, R. J. O’Keefe, B. F. Boyce, E. M. Schwarz. 2002. In vivo RANK signaling blockade using the receptor activator of NF-{kappa}B:Fc effectively prevents and ameliorates wear debris-induced osteolysis via osteoclast depletion without inhibiting osteogenesis. J. Bone Miner. Res. 17: 192-199. [Medline]
  15. Oyajobi, B. O., D. M. Anderson, K. Traianedes, P. J. Williams, T. Yoneda, G. R. Mundy. 2001. Therapeutic efficacy of a soluble receptor activator of nuclear factor {kappa}B-IgG Fc fusion protein in suppressing bone resorption and hypercalcemia in a model of humoral hypercalcemia of malignancy. Cancer Res. 61: 2572-2578. [Abstract/Free Full Text]
  16. Oyajobi, B. O., G. Franchin, P. J. Williams, D. Pulkrabek, A. Gupta, S. Munoz, B. Grubbs, M. Zhao, D. Chen, B. Sherry, G. R. Mundy. 2003. Dual effects of macrophage inflammatory protein-1{alpha} on osteolysis and tumor burden in the murine 5TGM1 model of myeloma bone disease. Blood 102: 311-319. [Abstract/Free Full Text]
  17. Pearse, R. N., E. M. Sordillo, S. Yaccoby, B. R. Wong, D. F. Liau, N. Colman, J. Michaeli, J. Epstein, Y. Choi. 2001. Multiple myeloma disrupts the TRANCE/osteoprotegerin cytokine axis to trigger bone destruction and promote tumor progression. Proc. Natl. Acad. Sci. USA 98: 11581-11586. [Abstract/Free Full Text]
  18. Yun, T. J., M. D. Tallquist, A. Aicher, K. L. Rafferty, A. J. Marshall, J. J. Moon, M. E. Ewings, M. Mohaupt, S. W. Herring, E. A. Clark. 2001. Osteoprotegerin, a crucial regulator of bone metabolism, also regulates B cell development and function. J. Immunol. 166: 1482-1491. [Abstract/Free Full Text]
  19. Stolina, M., J. Guo, R. Faggioni, H. Brown, G. Senaldi. 2003. Regulatory effects of osteoprotegerin on cellular and humoral immune responses. Clin. Immunol. 109: 347-354. [Medline]
  20. Gleichmann, E., I. Kimber, I. F. Purchase. 1989. Immunotoxicology: suppressive and stimulatory effects of drugs and environmental chemicals on the immune system: a discussion. Arch. Toxicol. 63: 257-273. [Medline]
  21. Burleson, G. R., H. Lebrec, Y. G. Yang, J. D. Ibanes, K. N. Pennington, L. S. Birnbaum. 1996. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on influenza virus host resistance in mice. Fundam. Appl. Toxicol. 29: 40-47. [Medline]
  22. Burleson, G. R.. 2000. Models of respiratory immunotoxicology and host resistance. Immunopharmacology 48: 315-318. [Medline]
  23. Lebrec, H., G. R. Burleson. 1994. Influenza virus host resistance models in mice and rats: utilization for immune function assessment and immunotoxicology. Toxicology 91: 179-188. [Medline]
  24. Morony, S., C. Capparelli, R. Lee, G. Shimamoto, T. Boone, D. L. Lacey, C. R. Dunstan. 1999. A chimeric form of osteoprotegerin inhibits hypercalcemia and bone resorption induced by IL-1beta, TNF-{alpha}, PTH, PTHrP, and 1,25(OH)2D3. J. Bone Miner. Res. 14: 1478-1485. [Medline]
  25. Morony, S., C. Capparelli, I. Sarosi, D. L. Lacey, C. R. Dunstan, P. J. Kostenuik. 2001. Osteoprotegerin inhibits osteolysis and decreases skeletal tumor burden in syngeneic and nude mouse models of experimental bone metastasis. Cancer Res. 61: 4432-4436. [Abstract/Free Full Text]
  26. Parfitt, A. M., M. K. Drezner, F. H. Glorieux, J. A. Kanis, H. Malluche, P. J. Meunier, S. M. Ott, R. R. Recker. 1987. Bone histomorphometry: standardization of nomenclature, symbols, and units: report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res. 2: 595-610. [Medline]
  27. Tamura, S., T. Kurata. 2004. Defense mechanisms against influenza virus infection in the respiratory tract mucosa. Jpn. J. Infect. Dis. 57: 236-247. [Medline]
  28. Boumpas, D. T., G. P. Chrousos, R. L. Wilder, T. R. Cupps, J. E. Balow. 1993. Glucocorticoid therapy for immune-mediated diseases: basic and clinical correlates. Ann. Intern. Med. 119: 1198-1208. [Abstract/Free Full Text]
  29. Markowitz, G. S., P. L. Fine, J. I. Stack, C. L. Kunis, J. Radhakrishnan, W. Palecki, J. Park, S. H. Nasr, S. Hoh, D. S. Siegel, V. D. D’Agati. 2003. Toxic acute tubular necrosis following treatment with zoledronate (Zometa). Kidney Int. 64: 281-289. [Medline]
  30. Body, J. J., I. Louviaux, J. C. Dumon. 2000. Decreased efficacy of bisphosphonates for recurrences of tumor-induced hypercalcemia. Support Care Cancer 8: 398-404. [Medline]
  31. Major, P. P., R. E. Coleman. 2001. Zoledronic acid in the treatment of hypercalcemia of malignancy: results of the international clinical development program. Semin. Oncol. 28: 17-24. [Medline]



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