OSTEOLITIC ACTIVITY OF OSTEOCLAST-LIKE CELLS DERIVED FROM PERIPHERAL BLOOD OF DIFFERENT SPECIES

Agnieszka Dziewicka¹, Natalia Stawińska², Iwona Ewa Kochanowska^1
1 Department of Experimental Therapy, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences
² Department of Periodontology, University of Medicine, Wroclaw, Poland

ABSTRACT

Osteolitic activity of human osteoclasts precursors derived from peripheral blood mononuclear cells (PBMC) was demonstrated several times by many investigators. However, there is insufficient knowledge about osteoclast’s precursors from PBMC of different species (mouse, rat, rabbit, bovine and guinea pig). Digestive activity of this cells manifests in their ability to bone collagen degradation in vitro in absence of stromal cells and without addition of growth factors, cytokines and steroids. The major markers generally used to identify osteoclasts are tartrate resistant acid phosphatase (TRAP) and calcitonin receptor (CTR) can be determinate by RT-PCR technique. C-telopeptides, markers of bone turnover caused by osteoclastic bone digestion, were determined with ELISA technique in supernatants. It was observed, that percentage of TRAP-positive cells in PBMC fraction differs in investigated species. Digestive properties of pre-osteoclasts manifested in amount of released C-telopeptides (CTx) depend on time of propagation and amount of propagated cells. TRAP and CTR genes expressions were detected in pre-osteoclasts of investigated species. Osteolitic properties of pre-osteoclasts derived from PBMC appear independently from addition of growth factors and cytokines and also without contact with initiating osteoclastogenesis cells like osteoblasts or stromal cells. High density cultures of pre-osteoclasts occur sufficient for successful bone digestion.

Key words: calcitonine receptor, C-telopeptides, osteoclast-like cells, peripheral blood mononuclear cells, pre-osteoclasts, tartrate resistant acid phosphatase.

INTRODUCTION

The aim of our investigation was to determinate potential osteolitic activity of osteoclasts precursors derived from peripheral blood mononuclear cells (PBMC) of different species (mouse, rat, rabbit, human, bovine and guinea pig).

Osteoclasts are large multinucleated cells, which play an important role in process of bone remodeling, which warrants bone homeostasis. In normal conditions resorption takes place after new bone formation. Cells that rebuild bone matrix in areas where bone has been resorbed by osteoclsts are called osteoblasts. [1].

There are evidences that osteoclast-like cells are present in PBMC population [2-4]. Many studies also shows that osteoclasts markers appears on cells derived from peripheral blood mononuclear cells in absence of stromal cells and without addition of growth factors, cytokines and steroids. These cells propagated in presence of bone slide are able to digest bone in vitro in the same manner as osteoclasts do [2,5]. Digestive activity of PBMC manifests in their ability to collagen type I degradation. C-telopeptides (CTx) are products of this process and their measurement allowed for specific bone resorption diagnosis. C-telopeptides can be determined in serum as well as in culture supernatants harvested from cells cultured in the presence of bone slides [6,7]. Recently, the routine way to evaluate the activity of osteoclasts was counting, under the microscope, the “pits” formed following bone fragment digestion. Pits assay enables to estimate osteoclasts activity by observation and counting areas of bone resorption. This technique is not only tedious and time consuming but highly inaccurate, because the recognition and identification of single pit sometimes raises doubts. Moreover, the mobility of osteoclasts complicates pits counting because of the pits overlapping and their localization on the ridges of bone fragments [8]. Therefore, we decided to substitute pit assay by C-telopeptide ELISA technique. This parameter directly depends on the amount of precursor cells and on their activity [7,9]. The amount of differentiated osteoclasts introduced into the culture can be estimated by their histochemical reaction showing one of osteoclastic marker i.e. tartrate resistant acid phosphatase (TRAP) [10]. This marker is required for osteoclastic bone resorption to decalcify matrix and it belongs to the class of metalloenzymes which catalyze the hydrolysis of phosphates esters and anhydrides in acidic reaction conditions [11, 12]. During osteoclast maturation several markers are expressed sequentionally. The first marker which appears on their surface is matrix metalloproteinase ninth (MMP9), the second one is tartrate-resistant acid phosphatase, next appear vitronectin receptor (VNR) and on the end of mononuclear stage, calcitonin receptor (CTR). Matrix metalloproteinase 9 are degrading all of bone matrix proteins, while TRAP decalcify bone environment. Vitronectin receptors allowed for adhesion to bone surface or cell to cell interaction and CTR binding with calctitonin. Therefore, we decided to determine not only TRAP but also calcitonine receptor with RT-PCR technique. Calcitonine receptor is phenotypic marker highly specific for osteoclast generation [13].

MATERIAL AND METHODS

PBMC isolation. Small blood samples were collected from animals accordingly to rules of Local Ethical Committee. The mixture of blood and PBS (1:1) was overlayed on Ficoll and centrifuged (2000 rpm/20 min in 4°C). The buffy coat interface of mononuclear cells was harvested and washed twice in PBS (1200 rpm/10 min in 4°C). Cells were suspended in 2 ml of Dulbecco’s Modified Eagle’s Medium (DMEM) with glutamine (0.2 M), 10% FBS (GIBCO BRL), streptomycin (10 mg·ml^-1), penicillin (10⁴ U·ml^-1) and vitamin D3 (5 µg·ml^-1). The number of peripheral blood mononuclear cells was determined in the cytometer.

Tartrate-resistant acid phosphatase (TRAP) specific staining of PBMC smears. PBMC suspended in DMEM (5 ľl) were smeared on slide and left to dry. Specific staining for TRAP reaction has been carried out using commercial set (“Leukocyte Acid Phosphatase Kit Reagents for The Cytologic Demonstration in Leucocytes from Blood, Bone Marrow and Tissue Touch Preparations” – SIGMA) according to manufacturer’s instruction. Percentage of TRAP-positive cells was estimated by counting under light microscope.

C-telopeptides measurements in supernatants. PBMC were cultured in 96 wells plates (Nunc) in Dulbecco’s Modified Eagle’s Medium (DMEM) with glutamine (0.2 M), 10% FBS (GIBCO BRL), streptomycin (10 mg·ml^-1), penicillin (10⁴ U·ml^-1) and vitamin D3 (5 ľg·ml^-1) in the presence of thin and small fragments of bone (area c-ca 25 mm2) obtained from frozen human dental bone. PBMC (0.5 × 10⁶, 1x10⁶ and 2 × 10⁶) suspended in 0.2 ml of medium were added into the wells and cultured for 8 days. Medium (150 µl) was changed after 24, 72, 120 and 168 hours. All removed supernatants were collected and frozen for C-telopeptide determination with ELISA technique (“CrossLaps for Culture” – Nordic Bioscience) accordingly to the manufacturer instruction.

Table 1. RT-PCR oligonucleotide primer sequences and expected sizes of product of amplification

RT-PCR. The amount and purity of total RNA isolated from 0.5-1×10⁶ of PBMC with TRIzol (SIGMA). Reagent was verified by spectrophotometric measurement. cDNA synthesized from 5 µg of total RNA using SUPERSCRIPT First-Strand Synthesis System (GIBCO BRL) did not exceeding 10% of PCR mixture volume. The sequences of oligonucleotide primers for this reaction were designed using Nucleotide – Nucleotide BLAST program which is available on-line on GenBank site (http://www.ncbi.nlm.nih.gov/BLAST/) basing on human sequences. The sequences of primers are listed in Table 1. The PCR reactions were carried out on a MJ Research PTC-200 Peltier Thermal Cycler with gradient block and consisted of initial denaturation at 94°C for 3 minutes followed by 30 amplification cycles as follows: 3-step cycling with denaturation at 94°C for 1 minute, annealing at 50°C for 1 minute, extension at 72°C for 1 minute, and final extension at 72°C for 10 minutes. Beta-actin was used as housekeeping. Product identity was confirmed by electrophoresis on a 1% agarose (GIBCO BRL) gel and visualized by ethidium bromide (SIGMA) staining under UV light.

RESULTS

PBMC isolation and tartrate-resistant acid phosphatase (TRAP) positive cells detection. The number of isolated PBMC determined in cytometer was different in all investigated species. The best isolation was from bovine blood while the worst was from guinea pig. Amount of PMBC obtained form 1 ml of guinea pig, mouse, rat, rabbit, bovine and human blood, respectively, was: 2.4 × 10⁵, 8.7 × 10⁵, 8.1 × 10⁵, 7.6 × 10⁵, 14.2 × 10⁶, and 7.7 × 10⁵. TRAP-positive osteoclasts precursors were observed as red/brown coloured cells under light microscope in 500 multiplication factor. The highest percentage of osteoclasts precursors was in guinea pig PBMC (32%), the lowest was in bovine PBMC (0.1%) population. The results obtained for all studied species are presented on Fig. 1. There is no correlation between amount of isolated PBMC and TRAP-positive cells contents.

Fig. 1. Percentage of TRAP-positive cells determined in PBMC population derived from all investigated species

C-telopeptides measurement in supernatants collected after PBMC propagation. Collagen’s I degradation products were measured with ELISA technique. The influence of time was determined for each species after 24, 72, 120 and 168 hours of propagation (Fig. 2). Apart from two species, mouse and human, releasing of C-telopeptides from digested bone to supernatant was similar and increase between 24 and 120 hours of propagation. Significant dependency between concentration of propagated cells and amount of released C-telopeptides was also observed (Fig. 3). PBMC from three species (human, bovine and rat) chosen for this experiment showed that optimal concentration of PBMC in culture is 1-2 × 10⁶.

Fig. 2. Influence of time on C-telopeptides’ amount obtained through bone degradation by PBMC (106 cells per well) and measured with ELISA

Fig. 3. Influence of propagated cells’ concentration on amount of released C-telopeptides

Fig. 4. TRAP and CTR expression studied by RT-PCR in osteoclast-like cells of different species

Comparison of TRAP and CTR expression levels in osteoclast-like cells from different species. To determinate expression of osteoclasts’ specific markers, TRAP and CTR, total RNA extracted from PBMC and transcribed onto cDNA was amplified in polymerase chain reaction using specific oligonucleotides. Expression of searching genes was detected in all investigated species (Fig. 4) apart from CTR gene in bovine PBMC. We didn’t obtain PCR product for bovine CTR expression and we couldn’t verified it because there were not sufficient data about this bovine gene in genome databases. Obtained amplicons were homogeneous and had proper length. It means that parameters of PCR like temperature of annealing and specificity of primers were correct and allowed for detection of TRAP and CTR genes expression.

DISCUSSION

There are only several reports referring behavior of osteoclasts’ precursors derived from PBMC of some animals describing conditions of human, mouse and rabbit pre-osteoclasts’ cultures supplemented by osteotropic factors and stromal cells. Additionally in these studies pre-osteoclasts phenotypes were determinate only by histochemical staining and not by molecular techniques [14,15,20].

In our studies osteolitic properties of PBMC derived from different species (human, guinea pig, mouse, rat, rabbit and bovine) were tested. Experiments of Matayoshi and Faust [2,5] gave us possibility to study in vitro differentiation of pre-osteoclasts and osteoclasts from PBMC. Our model differs from those proposed by Matayoshi and Faust. We decided to cultivate PBMC in absence of stromal cells, and threat them only with vitamin D3 (1.25(OH)₂ D3) not with others factors like M-CSF or interleukin 1 (IL-1) and interleukin 3 (IL-3). There are reports showing that pre-osteoclasts cultured in vitro in presence of bone slices are able to secrete cytokines (IL-1) and auto-stimulate to osteoclastogenesis [16]. Additional initiating osteoclastogenesis factor is cell to cell contact [8,17]. Therefore in our studies we used high density cultures which occur sufficient for successful bone digestion.

Osteoclasts as TRAP positive cells were identified by many investigators on the end of culture period [2, 5]. In our case we identified isolated cells possessing this marker before culture running, which assure us in presence of pre-osteoclast in PBMC population.

Measurement of C-telopeptides in urine and serum is known as a diagnostic method of estimating osteoclasts’ activity and bone turnover [7]. CTx can be also measured in culture medium, which is an alternative method for estimating of bone resorption. This assay is quantitative and more convenient than pits counting technique [18]. Using this technique we studied three parameters of PBMC derived pre-osteoclasts’ activity: (i) influence of culture time, (ii) influence of PBMC amount in the culture and (iii) comparison of osteolitic activity of PBMC from different species. There are no available reports in literature where these parameters were investigated. We showed that the highest concentration of CTx in culture media appear after 120-168 hours of propagation apart from two species, i.e. human and mouse. Pre-osteoclast cell from all investigated species released CTx in concentration range from 0.3 to 20 nM. This phenomenon depended not only on time of running culture but also on cells’ concentration. Our results proved that the highest CTx measurements were obtained where concentration of cell in culture was: 10⁶ for rat and bovine PBMC and 2 × 10⁶ for human PBMC. Comparison between species demonstrates distinct differences in osteolitic activity of investigated PBMC caused by differences in pre-osteoclasts contents in PBMC population. Presence of this specific biochemical marker of bone turnover in studied culture supernatants suggests that pre-osteoclasts in all cultures successfully developed in to forms capable to resorb bone matrix.

Expression of TRAP and CTR genes is characteristic for osteoclast differentiation process. The sequences of human TRAP and CTR primers, which we used in RT-PCR, were basing on sequences designed by Mbalaviele et al. [19]. RT-PCR technique has shown an activity of these genes in all species apart from CTR gene in bovine PBMC. We haven’t found reports referring expressions of TRAP and CTR on PBMC from species other than human. It is well known that sequences of these markers are conserved in many species and achieved 80-86% homology, what can be verified through gene homology analysis using NCBI BLAST. Therefore, for our examinations of investigated species, we decided to use primers designed basing on human TRAP and CTR nucleotide sequences. However, there are not available sequences in nucleotide database for guinea pig TRAP and CTR and bovine CTR gene. That’s why we used human primers in PCR. Expressions of both genes were detected in PBMC from all species excluded bovine CTR and length of obtained amplicons was adequate to length calculated during primers designing.

CONCLUSIONS

Using immunochemistry technique we proved the presence of osteoclasts in TRAP positive cells from PBMC population in all investigated species. RT-PCR technique allowed us to detect expression of mRNA not only for TRAP but also for another osteoclast-specific marker i.e.: CTR. Digestive activity of PBMC derived osteoclast manifested in their ability to collagen degradation was determined by measuring of C-telopeptide with ELISA technique. There were significant differences in osteolitic properties of those cells. Moreover, our results showed that PBMC derived osteoclast are able to differentiation and maturation in vitro in absence of stromal cells and without addition of growth factors, cytokines and steroids. However essential for differentiation and maturation processes occur time of propagation, amount of PBMC in the culture. Those conclusions let us to affirm that presumptions of our work, comparison of osteolitic activity of PBMC from different species, were achieved.

ACKNOWLEDGEMENTS

Funding was received from The Polish Committee for Scientific Research [00-529 Warsaw, Poland, Wspolna 1/3 St.] grant no. N401 128 31/3864.

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Accepted for print: 27.11.2007

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Agnieszka Dziewicka
Department of Experimental Therapy,
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences
Weigla 12 St., 53-114 Wroclaw, Poland
Phone: 071 370 99 17
email: dziewicka@iitd.pan.wroc.pl

Natalia Stawińska
Department of Periodontology,
University of Medicine, Wroclaw, Poland
Krakowska 26 St., 50-425 Wroclaw, Poland
Phone: 071 784 03 83
email: nstawin@wp.pl

Iwona Ewa Kochanowska
Department of Experimental Therapy,
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences
Weigla 12 St., 53-114 Wroclaw, Poland
Phone: 071 370 99 66
email: kochanie@iitd.pan.wroc.pl

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