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Poult Sci 2008. 87:2344-2349. doi:10.3382/ps.2008-00142
© 2008 Poultry Science Association
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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY

Chicken Receptor Activator of Nuclear Factor-{kappa}B Ligand Induces Formation of Chicken Osteoclasts from Bone Marrow Cells and also Directly Activates Mature Osteoclasts1

Y. Wang, J.-F. Hou and Z.-L. Zhou2

College of Veterinary Medicine, Nanjing Agricultural University, 210095, P. R. China

2 Corresponding author: zhouzl{at}njau.edu.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Receptor activator of nuclear factor-{kappa}B ligand (RANKL), which functions as a major determinant of osteoclast differentiation and activation, is a type II transmembrane protein and is expressed in osteoblasts-stromal cells. The aim of this study was to clarify the role of chicken RANKL (chRANKL) in chicken osteoclast differentiation and to determine its effect on mature chicken osteoclasts. In the present study, chRANKL protein was first cloned and expressed in Escherichia coli. We then treated chicken bone marrow cells with chRANKL protein and found that it induced the formation of chicken osteoclast-like multinucleated cells in a dose-dependent manner in the presence of human macrophage colony-stimulating factor. Moreover, the addition of chicken osteoprotegerin could block the effect of chRANKL with regard to osteoclast-like multi-nucleated cell formation and bone resorption. Using primary cultures of chicken osteoclasts on bone slices, we also found that bone resorption pits per cell increased with chRANKL concentration in a dose-dependent manner. The chRANKL-treated hens exhibited increased blood Ca++ levels within 2 h after injection, showing that chRANKL also activates osteoclasts in vivo. These results clearly indicate that the expressed protein is functional and may also be a critical factor for chicken osteoclastogenesis and bone resorption.

Key Words: chicken • receptor activator of nuclear factor-{kappa}B ligand • osteoclast • osteoclastogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Throughout adult life, normal bone metabolism involves a balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts (OC), resulting in a steady state of bone turnover in which there is no net change in overall bone mass. An imbalance between bone formation and bone resorption causes such metabolic bone diseases as osteopetrosis and osteoporosis (Roodman, 1996; Suda et al., 1997a). Bone fractures due to osteoporosis are also the most serious type of skeletal problem in modern laying hens (Whitehead, 2004). Osteoclasts are giant, multinucleated cells (MNC) and are the only cell type that can resorb bone (Lerner, 2000). Osteoclasts are formed by the fusion of OC precursors, which belong to the monocyte-macrophage lineage. Their differentiation in mammals is mainly triggered by receptor activator of nuclear factor-{kappa}B ligand (RANKL).

Receptor activator of nuclear factor-{kappa}B ligand, which is also known as OC differentiation factor, osteoprotegerin ligand, and tumor necrosis factor-related, activation-induced cytokine (Anderson et al., 1997; Wong et al., 1997; Lacey et al., 1998; Yasuda et al., 1998), is a recently reported member of the tumor necrosis factor superfamily. Receptor activator of nuclear factor-{kappa}B ligand, together with macrophage colony-stimulating factor, strongly induces the formation of OC-like cells (OLC) from mouse spleen cells (Yasuda et al., 1998) or human peripheral blood mononuclear cells (Matsuzaki et al., 1998) in the absence of osteoblasts-stromal cells. These lines of evidence indicate that RANKL plays a crucial role in mammalian osteoclastogenesis and bone resorption. In addition, osteoblasts-stromal cells also produce a factor called osteoprotegerin (OPG), which is a decoy receptor for RANKL. The discovery of the RANKL-OPG interaction provides a window into normal bone regulation and disease states.

However, the effect of chicken RANKL (chRANKL) on differentiation and activation of chicken OC is not clear. To investigate this question, we first cloned and expressed chRANKL protein in Escherichia coli (Wang et al., 2008) and then studied the ability of chRANKL to induce the formation of OLC from chicken bone marrow and its effect on mature chicken OC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Two 19-d-old and ten 18-d-old chicken embryos were obtained from China Animal Husbandry Industry Co. of Nanjing. The ISA caged layers were from Qinglongshan Husbandry Co. (Nanjing, China). Recombinant chRANKL protein and recombinant chicken OPG (chOPG) were obtained from our bone biology laboratory and have been described by Yao et al. (2006) and Wang et al. (2008). Dulbecco’s modified Eagle’s medium and {alpha}-minimum essential medium ({alpha}-MEM) were obtained from Gibco-BRL (Rockville, MD). Fetal bovine serum (FBS) was from Sijiqing Co. Ltd. (Hangzhou, China), and 24-well plates came from Bo Quan Sci & Tech. Co. Ltd. (Nanjing, China). The tartrate-resistant acid phosphatase (TRAP) staining kit came from Sigma-Aldrich (St. Louis, MO), and human macrophage colony-stimulating factor (M-CSF) was from R&D Systems (Minneapolis, MN). All other chemicals and reagents were of the greatest analytical grade.

Preparation of Bone Marrow Cells

Primary chicken bone marrow cells were isolated from the long bones of 2 chicken embryos (19 d of age) as described by Qian et al. (2006). Briefly, the embryos were first placed in 75% ethanol for 30 min, and then the tibias and femora were dissected free of adherent soft tissues, both epiphyses were cut off with scissors, and the marrow was vigorously flushed out with {alpha}-MEM. Chicken bone marrow cells were placed in a 75-cm2 flask and cultured in {alpha}-MEM containing 10% FBS. After a 2-h incubation, nonadherent cells were collected by centrifugation (639 x g) for 10 min. The isolated bone marrow cells were finally resuspended in {alpha}-MEM containing 10% FBS, 100 U/mL of penicillin, and 50 µ g/mL of streptomycin.

Effect of chRANKL and chOPG on OLC Formation and Bone Resorption

Isolated bone marrow cells were seeded on bovine bone slices (4 mm) or glass coverslips (6 mm) in 24-well plates. Chicken bone marrow cells (5 x 106 cells/mL) were cultured in {alpha}-MEM (1 mL/well) containing 10% FBS for 10 d in the presence of various concentrations of chRANKL, human M-CSF (hM-CSF), and chOPG (group A, 0 ng/mL of chRANKL + 0 ng/mL of hM-CSF; group B, 0 ng/mL of chRANKL + 25 ng/mL of hM-CSF; group C, 50 ng/mL of chRANKL + 0 ng/mL of hM-CSF; group D, 50 ng/mL of chRANKL + 25 ng/mL of hM-CSF; group E, 30 ng/mL of chRANKL + 25 ng/mL of hM-CSF; group F, 10 ng/mL of chRANKL + 25 ng/mL of hM-CSF; group G, 50 ng/mL of chRANKL + 25 ng/mL of hM-CSF + 100 ng/mL of chOPG). Cultures were fed every 3 d by replacing 0.5 mL of old medium with fresh medium with factors. All cultures were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37°C.

After culturing for 10 d, cells were fixed with glutaraldehyde and stained for TRAP as described previously (Suda et al., 1997b). The TRAP-positive cells containing 3 or more nuclei were scored as OLC. The total number of chicken OLC on each glass coverslip was counted using bright field optics with a Nikon Eclipse TS100 microscope (Nikon Inc., Melville, NY) and a 20x objective. The results were expressed as means ± SD for 7 cultures.

Mature OC Isolation and Culture

Primary chicken OC were prepared as described previously (Yao et al., 2007). Briefly, tibias and humeri were isolated from 10 chicken embryos (18 d of age) and cleaned of extraneous soft tissue without removing the bone ends, which were replete with OC (Colin-Osdoby et al., 2003). The marrow was removed from each bone by gripping the bone with alcohol-soaked tweezers, poking several small holes in each end of the bone using a syringe, and quickly flushing the marrow out by repeatedly inserting the tip of the syringe filled with Hanks’ buffered salt solution (HBSS, pH 7.2) into the end of the bone. After all of the marrow was extruded, the bones were placed in a clean dish on ice, and each bone was split and submersed in HBSS. The split bones were transferred to polypropylene tubes containing HBSS, shaken vigorously for 30 s, and the suspended cells (from the inside surface of bones) were collected sequentially. The cell pellet was resuspended in {alpha}-MEM with 15% FBS, 100 U/mL of penicillin, and 50 µ g/mL of streptomycin. Cell suspensions were re-plated at 5 x 105 cells per well in 24-well dishes containing glass coverslips or bovine bone slices. Nonadherent cells were washed off after 2 h, and the medium was changed every 48 h thereafter. The adherent cells were grown for an additional 6 d, during which time chRANKL was added at different concentrations (2, 6, and 10 ng/mL).

Bone Resorption Measurements

After fixation with 0.25% glutaraldehyde, bone slices were stained for TRAP. Mature OC were defined as highly TRAP-positive cells containing 3 or more nuclei. The total number of chicken OC on each bone slice was counted using bright field optics with a Nikon Eclipse 800 upright microscope and a 20x objective. After counting, the OC were removed using 50 mM NH4OH and brief sonication. The resorption lacunae on the same bone slices were then visualized by toluidine blue staining. An individual resorption event was distinguished by a dark border of toluidine blue stain surrounding an excavation, according to Burgess et al. (1999). The data presented here were recorded for each resorption event separately; often several events were apparent in what is classically called a resorption pit. The data were expressed as the average number of pits per OC (mean ± SD).

Scanning Electron Microscopy

After toluidine blue staining, the bovine bone slice was sonicated in water to remove the stain and any residue. It was dehydrated through a graded ethanol series and left in 100% ethanol overnight. After air drying, the slices were placed in a vacuum desiccator for several hours before being mounted on scanning electron microscope (EM) stubs. The mounted slices were sputter-coated with 30 nm of gold-palladium. The specimens were examined on a Philips 505 scanning EM (Philips, Eindhoven, the Netherlands) at 25 kV with a working distance of 20 mm. Resorption lacunae from each slice were identified with scanning EM, and a representative example was selected for photography at 460x magnification.

In Vivo Caged Layer Experiment

Fifty 58-wk-old ISA caged layers were divided into 5 groups (10 hens per group) and were fed a low-calcium diet for 48 h before receiving varying doses of chRANKL by intramuscular injection in a PBS carrier, or PBS alone as control. Blood samples (5 mL per hen from wing vein) were obtained 2 h after injection for determination of ionized calcium. Blood-ionized calcium levels were then determined using an autoanalyzer (Selectra-E-plus, Vital Scientific, Dieren, the Netherlands).

Statistical Analysis

For control and treatment groups, the means and SD were calculated with SPSS 13.0 (SPSS Inc., Chicago, IL). Values of calculated means were compared among groups using 1-way ANOVA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of chRANKL on OLC Formation

After a 10-d incubation, no TRAP-positive MNC were observed on the glass coverslips when chicken bone marrow cells were cultured in the absence of chRANKL and hM-CSF (Figure 1AGo). On the other hand, a few TRAP-positive MNC were found on the glass cover-slips in the culture treated with chRANKL and hM-CSF (Figure 1B, 1C, 1DGo). The number of TRAP-positive MNC formed in the presence of 50 ng/mL of chRANKL was more than that formed at 30 and 10 ng/mL, respectively (P < 0.05; Figure 2Go). The addition of chOPG (100 ng/mL) to the culture resulted in strong inhibition of OLC formation (Figure 1EGo).


Figure 1
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  		Figure 1. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNC) in cultured chicken bone marrow cells. A. No TRAP-positive MNC in the absence of chicken receptor activator of nuclear factor-{kappa}b ligand (chRANKL) or human macrophage colony-stimulating factor (hM-CSF). B. A few TRAP-positive MNC (arrows) can be observed in the culture with chRANKL (10 ng/mL) and hM-CSF (25 ng/mL). C. Culture with chRANKL (30 ng/mL) and hM-CSF (25 ng/mL). D. Culture with chRANKL (50 ng/mL) and hM-CSF(25 ng/mL). E. Culture with chRANKL (10 ng/mL), hM-CSF (25 ng/mL), and chicken osteoprotegerin (100 ng/mL) 200x.

 

Figure 2
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  		Figure 2. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells after 10 d of incubation in response to chicken receptor activator of nuclear factor-{kappa}b ligand (RANKL), human macrophage colony-stimulating factor (M-CSF), and chicken osteoprotegerin (OPG), showing a dose-dependent effect.

 
On d 14, no resorption pits were observed on the bovine bone slices when marrow cells were cultured in the absence of chRANKL or hM-CSF. Numerous resorption pits were formed on bovine bone slices when cultured in the presence of chRANKL and hM-CSF (Figure 3A, 3BGo), and bone resorption was completely inhibited when chOPG (100 ng/mL) was added to the culture (data not shown).


Figure 3
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  		Figure 3. Resorption pits (arrows) on bone slices after 14 d. A. Formation of resorption lacunae confirmed by toluidine blue staining, 250x. B. Resorption lacunae on the bone slice were confirmed by scanning electron microscopy, 460x.

 
chRANKL Increases the Number of Pits Per OC Cell

We found that the effect of chRANKL on resorption index was dose-dependent, because the index increased from 1.2 at 2 ng/mL up to 3.08 at 10 ng/mL (P < 0.05; Figure 4Go). This result clearly showed that chRANKL can also promote mature OC activity.


Figure 4
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  		Figure 4. Relationship between chicken receptor activator of nuclear factor-{kappa}b ligand (RANKL) and number of pits per cell (P < 0.05).

 
chRANKL Stimulates a Rapid Onset of Hypercalcemia in Hens

Randomized groups of hens (n = 10) were injected intramuscularly with chRANKL at increasing concentrations (Figure 5Go). After 2 h, the levels of ionized calcium in the blood were determined as a measurement of OC activation, and the results are shown in Figure 5Go. Chicken RANKL increased whole blood ionized calcium levels dose-dependently with significant increases seen at doses of 0.05, 0.1, or 0.5 mg/kg (P < 0.05).


Figure 5
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  		Figure 5. Relationship between chicken receptor activator of nuclear factor-{kappa}b ligand (RANKL) and blood-ionized calcium levels in chickens. At doses of 0.05, 0.1, and 0.5 mg/kg, statistically significant increases in blood ionized calcium were detected,

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The goal of this study was to use a functional recombinant chRANKL protein (Wang et al., 2008) to investigate the role of chRANKL in OC activation. We demonstrated that chRANKL was capable of inducing OLC formation as well as bone resorption in chicken bone marrow cell culture. Chicken OC have been frequently used as a model to study bone resorption in vitro. However, they are considered different from mammalian OC, because the calcitonin receptor, a functional marker of mammalian OC, has never been detected on these cells; the responsiveness of chicken OC to chRANKL and calcitonin is still debatable (Sexton et al., 1999). Boissy et al. (2001) have supported the notion that chicken OC are closer to mammalian OC than previously thought. The present study showed that chicken RANKL could induce the formation of MNC, which were shown to be TRAP-positive cells, and exhibited evidence of resorption pit formation on bone slices. Thus, chRANKL-induced MNC derived from cultured chicken bone marrow cells were indeed OLC according to the definition of OC (Suda et al., 1997; Kartsogiannis and Ng, 2004). It is also known that OPG plays a key role in maintaining normal bone metabolism by inhibiting the action of RANKL in mammals (Suda et al., 1999). The TRAP-positive MNC could be seen in cultures treated with 50 ng/mL of chRANKL and 25 ng/mL of hM-CSF, but fewer TRAP-positive MNC and no resorption pits could be observed after the addition of chOPG. This result could indicate that chOPG blocks the effects of chRANKL on OC formation. Our data also show that chRANKL activated mature OC to resorb bone in vitro in a dose-dependent manner. Furthermore, chRANKL caused hypercalcemia within 2 h in vivo [similar to results in mice (Burgess et al., 1999)], possibly due to the activation of preformed OC (Lacey et al., 1998). We found that chickens on a low-calcium diet for 48 h showed a dose-dependent elevation in blood-ionized calcium in response to chRANKL, thus ruling out gut absorption as the source of calcium. Given the rapid activation of OC in vitro by chRANKL by the bone resorption index shown here, it seems that OC activation could be involved in vivo as well.

Suda et al. (1999) had confirmed that M-CSF is indispensable to both the proliferation phase and the differentiation phase of OC development. More recently, Fuller et al. (1998) reported that M-CSF prolonged the survival of mature OC, whereas in the absence of M-CSF, the number of OC was strikingly decreased. This study confirmed the role of M-CSF in chicken OC differentiation. No TRAP-positive OC could be seen in the presence of M-CSF alone, but TRAP-positive OC and resorption pits were observed in the presence of chRANKL and hM-CSF.

In summary, the present study is the first to demonstrate that chRANKL and M-CSF are 2 important factors required for inducing osteoclastogenesis in chickens, similar to that observed for mammals. Chicken RANKL and M-CSF could induce OLC formation and bone resorption in chicken marrow cell culture, and this could serve as a model to study osteoclastogenesis in chickens and investigate the effects of other factors on chicken OC differentiation and activation. The present study also indicates the possibility that RANKL and OPG may be targeted for the treatment of chicken bone metabolic diseases such as osteoporosis.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the National Natural Science Foundation of China (30671546), Education Ministry Major Program of China (107059), and Natural Science Foundation of Jiangsu Province (BK2004099).


   FOOTNOTES
 
1 The nucleotide sequence of chicken receptor activator of nuclear factor-{kappa}B ligand we cloned has been sent to GenBank, and the accession number is EF379383. Back

Received for publication April 3, 2008. Accepted for publication June 25, 2008.


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Colin-Osdoby, P., F. Anderson, and P. Osdoby. 2003. Primary isolation and culture of chicken osteoclasts. Page 71 in Bone Research Protocols. M. H. Helfrich and S. H. Ralston, ed. Humana Press Inc., Totowa, NJ.

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