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Clinical Chemistry 46: 1751-1754, 2000;
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(Clinical Chemistry. 2000;46:1751-1754.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Rapid Screening Method for Osteoclast Differentiation in Vitro That Measures Tartrate-resistant Acid Phosphatase 5b Activity Secreted into the Culture Medium

Sari L. Alatalo1,a, Jussi M. Halleen1, Teuvo A. Hentunen1,2, Jukka Mönkkönen3 and H. Kalervo Väänänen1

1 Institute of Biomedicine, Department of Anatomy, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland.

2 Cell Test Turku Ltd, FIN-20520 Turku, Finland.

3 Department of Pharmaceutics, University of Kuopio, FIN-70210 Kuopio, Finland.
a Author for correspondence. Fax 358-2-333 7352; e-mail saleal{at}utu.fi


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Osteoclasts secrete tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) 5b into the circulation. We studied the release of TRAP 5b from osteoclasts using a mouse in vitro osteoclast differentiation assay.

Methods: We developed and characterized a polyclonal antiserum in rabbits, using purified human osteoclastic TRAP 5b as antigen. The antiserum was specific for TRAP in Western analysis of mouse osteoclast culture medium and was used to develop an immunoassay. We cultured mouse bone marrow-derived osteoclast precursor cells for 3–7 days with or without clodronate in the presence of vitamin D and analyzed the number of osteoclasts formed and the amount of TRAP 5b activity released into the culture medium.

Results: TRAP 5b activity was not secreted from osteoclast precursor cells. Addition of clodronate-containing liposomes decreased in a dose-dependent manner the number of osteoclasts and TRAP 5b activity released in 6-day cultures. The amount of TRAP 5b activity in the medium detected by the immunoassay correlated significantly with the number of osteoclasts formed (r = 0.94; P <0.0001; n = 120).

Conclusions: The TRAP 5b immunoassay can be used to replace the laborious and time-consuming microscopic counting of osteoclasts in the osteoclast differentiation assay and to test the effects of potential therapeutic agents on osteoclast differentiation, enabling fast screening of large amounts of potential therapeutic agents.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Osteoclast precursors in mouse bone marrow can be induced to form multinucleated osteoclast-like cells in the presence of the active metabolite of vitamin D3, 1,25-dihydroxycholecalciferol [1,25(OH)2D3]. The multinucleated osteoclast-like cells form pits on bone or dentine slices (1)(2)(3), express high concentrations of tartrate-resistant acid phosphatase (TRAP; EC 3.1.3.2) and calcitonin receptors (3)(4), and respond to calcitonin and prostaglandin E2 (1)(5). Thus, the system is ideal for studying potential therapeutic agents targeted at the inhibition of osteoclastogenesis. At present, the number of osteoclasts formed is determined by counting the number of TRAP-positive multinucleated osteoclast-like cells containing at least three nuclei (1).

Osteoclasts express high amounts of TRAP, also known as type 5 acid phosphatase. TRAP has been used for years as a marker of osteoclasts in bone (6). Osteoclasts secrete TRAP into the circulation, suggesting that serum TRAP may be a useful resorption marker. Two forms of TRAP, known as 5a and 5b, are found in the circulation; 5b is derived from osteoclasts and 5a from some other source (7)(8). Our previous results showed that an immunoassay specific for TRAP 5b is a useful method to monitor antiresorptive treatment, whereas total serum TRAP cannot be used in that purpose (8).

We studied the secretion of TRAP 5b from osteoclasts in mouse bone marrow cell cultures, using a novel TRAP 5b-specific immunoassay, and found that the immunoassay is a fast and simple method to determine the number of osteoclasts formed in the in vitro osteoclast differentiation assay. Thus, the immunoassay can be used to test the effects of potential inhibitors of osteoclastogenesis, enabling fast and reliable screening of large numbers of potential therapeutic agents.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
bone marrow cell cultures
Bone marrow cells from the femora and tibia of 8-week-old mice were cultured for 3–7 days on plastic in the presence of 10 nmol/L 1,25(OH)2D3 to induce osteoclast formation (9)(10). Clodronate-containing liposomes were prepared as described (11), and the effect of 10-5–10-8 mol/L clodronate was studied by the addition of liposomes into the culture medium (12). Cells were fixed with 30 g/L paraformaldehyde and stained for TRAP with a histochemical method (Sigma). TRAP-positive cells containing three or more nuclei were counted with a light microscope (Leica Aristoplan).

TRAP 5b IMMUNOASSAY
TRAP was purified from human osteoclasts as described (13), and the purified enzyme was used as antigen to develop a polyclonal TRAP antiserum in rabbits. A 1:1000 dilution of the antiserum was used. Western analysis was performed as described (8). In the immunoassay, the antiserum was incubated on anti-rabbit IgG-coated microtiter plates (EG & G Wallac) for 1 h. Culture medium (200 µL) was incubated in the wells for 1 h, and bound enzyme activity was detected using 8 mmol/L 4-nitrophenyl phosphate as substrate in 0.1 mol/L sodium acetate buffer for 2 h at 37 °C. The enzyme reactions were terminated by the addition of 25 µL of 0.32 mol/L NaOH to the wells, and the absorbance at 405 nm (A405) was measured with a model 2 Victor instrument (EG & G Wallac).

characterization of the immunoassay
We tested the stability of TRAP in mouse bone marrow cell culture medium by storing medium samples for up to 7 days at 25 and 4 °C. Recovery was determined by adding various amounts of human osteoclastic TRAP into the culture medium and calculating the amount detected. The intraassay CV was determined as the mean value obtained from five aliquots of three pooled medium samples simultaneously, and the interassay CV was determined from the same samples measured on 6 consecutive days.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
characterization of the antiserum and the immunoassay
The polyclonal antiserum bound the maximal amount of TRAP when diluted up to 1:1000 (Fig. 1 A). In Western analysis, the antiserum detected purified human osteoclastic TRAP (Fig. 1B , lane 1), and it was specific for TRAP in mouse osteoclast culture medium (Fig. 1B , lane 2) and in human bone homogenate (Fig. 1B , lanes 3 and 4). A typical calibration curve of an immunoassay using the antiserum is shown in Fig. 1C . The recovery of various amounts of purified human osteoclastic TRAP from mouse osteoclast cell culture medium (collected after a 6-day culture) was 104.6% ± 3.1%. The intra- and interassay CVs were 1.6% and 5.1%, respectively. TRAP activity was stable in the culture medium (no statistically significant change from baseline) for 8 h at 25 °C and for 3 days at 4 °C. After incubation for 7 days at 4 and 25 °C, 76% and 30% of the initial TRAP activity was detected, respectively.



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  		Figure 1. Characterization of the polyclonal antiserum and the immunoassay.

(A), determination of the antiserum titer, using purified human osteoclastic TRAP. (B), Western analysis. The arrow indicates the 32-kDa TRAP. Lane 1, purified human osteoclastic TRAP (10 µg); lane 2, concentrated mouse bone marrow cell culture medium; lane 3, concentrated fraction containing proteins of human bone homogenate that were bound to the cation-exchange column; lane 4, concentrated fraction containing human bone homogenate that did not bind to the cation-exchange column. (C), calibration curve of the immunoassay. Purified human osteoclastic TRAP was used as calibrator. One unit of enzyme activity hydrolyzes 1 µmol of 4-nitrophenyl phosphate per minute at 37°C.

medium TRAP 5b as an index of osteoclast number
Both the number of osteoclasts formed and the amount of TRAP 5b activity released into the culture medium were significantly (P <0.001) increased with increased culture time in mouse bone marrow cell cultures (Fig. 2 A). Clodronate decreased the number of osteoclasts and TRAP 5b activity released into the culture medium in a dose-dependent manner (Fig. 2B ). Osteoclast formation correlated significantly with the amount of TRAP 5b released into the culture medium (r = 0.94; P <0.0001; n = 120), as shown in Fig. 2C .



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  		Figure 2. TRAP 5b activity in medium as an index of osteoclast number in mouse bone marrow cell culture.

(A), relative TRAP 5b activity in medium and osteoclast number after culture for 3–7 days in the presence of 1,25(OH)2D3. The mean values of 7-day cultures were given the value 1.0. (B), effect of 10-5–10-8 mol/L clodronate on osteoclast number and TRAP 5b activity in 6-day cultures. The mean values of the baseline group were given the value 1.0. A 6-day culture without 1,25(OH)2D3 was included as negative control. (C), correlation of TRAP 5b activity in medium with the number of osteoclasts formed. The combined data of the individual cultures in the groups shown in panels A and B were used in panel C.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show here that the amount of TRAP 5b activity released into the culture medium in bone marrow-derived osteoclast precursor cell cultures has a significant correlation with the number of osteoclasts formed. Our TRAP 5b immunoassay can be used to replace the laborious and time-consuming microscopic counting of osteoclasts. For 120 wells, it takes ~1 week to count osteoclasts and ~6 h, of which 4 h are for incubations, to determine the TRAP 5b activity. Thus, with this new method, a study that previously took 1 week can now be performed with ~2 h of work in the laboratory.

Clodronate is used as a therapeutic agent in osteoporosis. It inhibits bone resorption by binding to bone surface, from which it is internalized by resorbing osteoclasts, leading to osteoclast apoptosis (12). When clodronate is added in liposomes, it can also enter osteoclast precursor cells and inhibit osteoclast formation. We used clodronate-containing liposomes to study the effect of inhibition of osteoclastogenesis on the amount of TRAP 5b in medium, and noticed a strong correlation of osteoclast number with the amount of TRAP 5b in the medium after clodronate treatment.

We found no correlation of TRAP 5b activity in medium with the number of mononuclear TRAP-positive cells in the cultures, suggesting that mononuclear osteoclast precursor cells would not secrete TRAP 5b. For example, at day 4, when there were 300–500 mononuclear TRAP-positive cells in the cultures (data not shown) and only 15–30 osteoclasts, TRAP 5b activity in the medium was very low.

Our data indicate that mature osteoclasts secrete TRAP 5b constitutively into the culture medium. This suggests that the amount of TRAP 5b released into the circulation may indicate the number of mature osteoclasts in bone rather than their resorbing activity. Increased bone resorption is associated with increased osteoclast number in several bone diseases, such as osteoporosis, Paget disease, hyperparathyroidism, and cancer bone metastases. In addition, most antiresorptive drugs such as estrogen, selective estrogen receptor modulators, and bisphosphonates inhibit osteoclastogenesis. Thus, if TRAP 5b is a marker of osteoclast number, it may be a useful marker in the diagnosis of bone diseases and in monitoring antiresorptive treatment.

Is TRAP 5b released from osteoclasts constitutively in vivo? Resorbing osteoclasts are polarized and transport matrix degradation products in transcytotic vesicles from the resorption lacuna to a functional secretory domain in the basolateral membrane (14)(15)(16). In polarized osteoclasts, TRAP is targeted into the transcytotic vesicles and secreted through functional secretory domain (17). In the in vitro osteoclast differentiation assay, the cells are cultured on plastic. Thus, the cells may not be polarized, and TRAP 5b may be targeted directly into the basolateral membrane and secreted. Mice overexpressing TRAP have normal amounts of osteoclasts, increased bone resorption, and increased serum TRAP (18), suggesting that in vivo, TRAP secretion would be associated with the resorbing activity of osteoclasts.

Whatever the mechanism of TRAP secretion is in vivo, we have demonstrated that TRAP 5b is secreted constitutively from osteoclasts when they are cultured on plastic. The number of osteoclasts shows a significant correlation with the TRAP 5b activity in the culture medium. The TRAP 5b immunoassay could be a useful method in experiments studying the effects of potential therapeutic agents on osteoclastogenesis, enabling fast and reliable screening of large numbers of potential therapeutic agents.


   Acknowledgments
 
This work was supported by the Graduate School of Musculo-Skeletal Diseases (TULES), the Academy of Finland, and the State Technology Development Center of Finland (TEKES).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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