Med Mol Morphol DOI 10.1007/s00795-014-0094-8
ORIGINAL PAPER
Expression of interleukin-34 and colony stimulating factor-1 in the stimulated periodontal ligament cells with tumor necrosis factor-a Mutsuki Kawabe • Hideki Ohyama • Nahoko Kato-Kogoe Naoko Yamada • Koji Yamanegi • Hiroshi Nishiura • Hirotugu Hirano • Hiromitsu Kishimoto • Keiji Nakasho
•
Received: 14 November 2014 / Accepted: 17 December 2014 Ó The Japanese Society for Clinical Molecular Morphology 2014
Abstract Tumor necrosis factor-a (TNF-a) directly and indirectly plays a crucial role in osteoclastogenesis. However, the indirect effects of TNF-a on colony-stimulating factor-1 receptor (CSF-1R)-mediated osteoclastogenesis achieved via periodontal ligament (PDL) cells are not fully understood. We herein examined the potency of osteoclast differentiation and maturation induced by fivefold supernatants in the stimulated human PDL cells with a physiologically high concentration (10 ng/mL) of recombinant TNF-a to human peripheral blood monocytes/macrophages in the simultaneous presence of the receptor activator of nuclear factor kappa-B ligand. The number of tartrateresistant acid phosphatase-positive cells with multiple nuclei, but not those with a single nucleus, was decreased by approximately 50 % by neutralization with rabbit IgG against either interleukin-34 (IL-34) or CSF-1. Small and large amounts of IL34 and CSF1 transcripts were measured in the stimulated PDL cells using real-time polymerase chain reaction. The corresponding amounts of proteins to IL34 and CSF1 transcripts were observed in the stimulated PDL cells on immunohistochemical staining or Western blotting. Moreover, 0.13 ng/mL of IL-34 and 5.0 ng/mL of CSF-1 were measured in the supernatants of the stimulated M. Kawabe (&) H. Kishimoto Department of Oral and Maxillofacial Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Japan e-mail:
[email protected] H. Ohyama N. Kato-Kogoe N. Yamada K. Yamanegi H. Nishiura K. Nakasho Department of Pathology, Hyogo College of Medicine, Nishinomiya 663-8501, Japan H. Hirano Department of Laboratory Medicine, Toneyama National Hospital, Osaka 560-8552, Japan
PDL cells using an enzyme-linked immunosorbent assay. IL-34 derived from the stimulated PDL cells with TNF-a appeared to synergistically function with CSF-1 in the CSF-1R-mediated maturation of osteoclastogenesis. Keywords Interleukin-34 Colony-stimulating factor-1 Osteoclastogenesis Periodontal ligament cells Tumor necrosis factor-a
Introduction Interleukin-34 (IL-34) has been identified to be an alternative ligand of colony-stimulating factor-1 receptor (CSF1R) to CSF-1 by screening a comprehensive human protein library using a human monocyte viability assay [1]. The protein homology between human IL-34 (hIL-34, GI: 20987450) and CSF-1 (hCSF-1, GI: 18644893) is under 5 % (blastp suite-2 sequences, BLAST, NCBI), while that of IL-34 between human and mouse (mIL-34, GI: 205360952) is over 70 %. In contrast to the monomer form of mCSF-1, the dimer form of mIL-34 makes a mCSF-1R bridge [2]. The flexible link between the N-terminal of two immunoglobulin-like domains of mCSF-1R allows for different binding positions between mIL-34 and mCSF-1 [3]. The binding affinity of mIL-34 against mCSF-1R is lower than that of mCSF-1 [4]. Therefore, the IL-34induced mCSF-1R-mediated downstream pathway and its consequent specific transcriptional profiles are believed to be slightly different from those of CSF-1 [5]. The degree of differentiation of macrophages, microglia and osteoclasts in CSF-1R-deficient mice is weaker than that observed in CSF-1-deficient mice [6]. In particular, the number of microglia is significantly low in IL-34-deficient mice compared with that noted in CSF-1-deficient mice. In
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the embryonic brain at E11.5, the gene expression of IL34 is high to that of CSF1 [4]. Moreover, the area of the gene expression of IL34 in the mouse brain does not overlap with that of the CSF1 expression. Conversely, IL-34 has been suggested to similarly regulate the differentiation of osteoclasts and M2 macrophages to CSF-1 via CSF-1R, which subsequently promotes cell proliferation and tissue repair [7, 8]. Tumor necrosis factor-a (TNF-a) indirectly functions as a producer of various transcription factors, including nuclear factor-kappa B (NF-kB), activator protein-1 (AP-1), interferon regulatory transcription factor-1 (IRF-1) and NF-GMa, to induce the onset of CSF-1R-mediated osteoclastogenesis in periodontal tissues [9–12]. The gingival epithelium, gingival fibroblasts and periodontal ligament (PDL) cells in periodontal tissues secrete CSF-1 to initiate osteoclastogenesis in a transcription factor-dependent manner [13]. Conversely, the IL-34 derived from gingival fibroblasts has recently been reported to function as a substitute for CSF-1 in the simultaneous presence of the receptor activator of nuclear factor kappa-B ligand (RANKL) in the induction of osteoclastogenesis [14]. The protein expression of mIL-34 is lower than that of mCSF-1 in gingival fibroblasts [15]. However, the mIL-34 dimer facilitates the creation of the mCSF-1R bridge [2]. These observations suggest the synergistic effects of IL-34 in association with CSF-1 on CSF1R-mediated osteoclastogenesis. In addition to gingival fibroblasts, periodontal ligament (PDL) cells are known to produce CSF-1 [16]. To study the synergistic function of IL-34 derived from PDL cells acting with CSF-1 on CSF-1R-mediated osteoclastogenesis in the simultaneous presence of RANKL and TNF-a, we herein prepared supernatants of human PDL cells stimulated with a physiologically high concentration of TNF-a of 10 ng/mL.
Materials and methods Cell culture Periodontal ligament cells were isolated from young patients undergoing orthodontic treatment according to the method of Nishimura (n = 3) [17]. Briefly, after washing the extracted teeth in phosphate-buffered saline (PBS) containing the following final concentrations of penicillin (100 lg/mL; Sigma–Aldrich, St. Louis, MO, USA), streptomycin (100 lg/mL; Sigma–Aldrich), neomycin (200 lg/mL; Sigma–Aldrich) and amphotericin B (0.5 lg/ mL; Sigma–Aldrich) three times, the PDL tissues in the middle third of the tooth root were transferred to 10 mL of PBS containing the following final concentrations of dispase-II (16 mg/mL; Sigma–Aldrich) and collagenase type-I (12 mg/mL; Sigma–Aldrich) in 15-mL tubes and rotated
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for 60 min at 37 °C. The PDL cells were then transferred to 10 mL of a-minimum essential medium (Invitrogen, Carlsbad, CA, USA) containing 10 % fetal bovine serum (FBS; Bioindustry Division, Itabashi-ku, Tokyo, Japan), penicillin and streptomycin (the culture medium) in U10cm culture dishes. After passaging five or six times in the culture medium, the PDL cells were stocked in nitro oxygen liquids until use for the experiments. Osteoclastogenesis Supernatants of the stimulated PDL cells with TNF-a (10 ng/mL; PeproTech Inc., Rocky Hill, NJ, USA) for 4 days in the culture medium without FBS (the differentiation medium) were pretreated with a vehicle buffer, control rabbit IgG, anti-IL-34 rabbit IgG and/or anti-CSF-1 mouse IgG2 (100 ng/mL; R&D systems, Koto-ku, Tokyo, Japan). After depleting the IL-34 and/or CSF-1 trapped by each antibody using Dynabeads Protein G (Applied Biosystems, Inc., Foster, CA, USA), the amount of IL-34 or CSF-1 proteins in the supernatants was measured using an enzymelinked immunosorbent assay according to the manufacturer’s protocol (R&D systems). After treating the cells with Amicon (Millipore Corporation, Billerica, MA, USA), we confirmed the minimum osteoclastogenesis potency of the fivefold supernatants in our experimental setting. Briefly, peripheral blood monocytes/macrophages obtained from healthy volunteers were isolated using the Human Monocyte Enrichment Kit (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer’s protocol. The monocytes/macrophages were plated at 2 9 104 cells/well in 96-well culture plates (BD, Minato-ku, Tokyo, Japan) and maintained for 12 days in the differentiation medium containing the fivefold supernatants and RANKL (20 ng/mL, Bioindustry Division). Following washing and fixing, the cells were stained with tartrateresistant acid phosphatase (TRAP) in a leukocyte acid phosphatase kit (Sigma, St. Louis, MO, USA). After the staining procedure, the cells were photographed with a microscope (TE300-HM-2, Nikon, Tokyo, Japan) equipped with a charge-coupled device (CCD) digital camera (Nikon) and software program. We counted the number of cells per well on the images using an image analysis software package (ImageJ 1.48, Bethesda, MD, USA) and determined the number of multinucleated TRAP? cells with more than three nuclei, defined as multinucleated osteoclast-like cells, per well. Gene expression The PDL cells were stimulated in the differentiation medium on 24-well culture plates (BD) for 48 h with 10 ng/mL of TNF-a. cDNA acquired from the total RNA
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of the stimulated PDL cells using the High Capacity RNAto-cDNA Kit (Invitrogen) was prepared as a template. Following polymerase chain reaction (PCR) with the specific primer pairs (Table 1) in the SYBR Green PCR Master Mix (Invitrogen) (7500-01; Applied Biosystems Inc.), the products were quantified in a real-time PCR machine.
Results Potency of osteoclastogenesis induced by the supernatants of the stimulated PDL cells with TNF-a
Immunohistochemical staining The PDL cells were stimulated in the differentiation medium on the chamber slides (BD) for 96 h with 10 ng/ mL of TNF-a. After fixing and quenching the endogenous peroxidase activity, the PDL cells were stained with antiIL-34 (R&D systems) or anti-CSF-1 (Genzyme, Cambridge, MA, USA) rabbit IgG for 60 min and Dako EnVisionTM ? Dual Link System-HRP (Dako, Bunkyo-ku, Tokyo, Japan) for another 15 min at room temperature. The protein expression was visualized using a 3,30 -diaminobenzidine (DAB) tablet (Dako). The cells were subsequently photographed with a microscope equipped with a CCD digital camera, and the protein density on the pathological photographs was measured using the ImageJ 1.48 program. Western blotting The PDL cells were stimulated in the differentiation medium on U10-cm culture dishes for 4 days with 10 ng/ mL of TNF-a. The lysates (10 lg/lane) of the stimulated PDL cell were applied to 4–12 % sodium dodecyl sulfate polyacrylamide gel (Invitrogen), and the proteins transferred on polyvinylidene difluoride (PVDF) membranes (Invitrogen) were reacted with the primary antibodies and horseradish peroxidase conjugated secondary antibodies (Santa Cruz Biotechnology; Dallas, TX, USA). Proteins were detected using Chemi-Lumi One (Nakarai Tesque; Deguchi, Suita, Osaka), and the density was measured using the ImageJ 1.48 program. Statistical analysis The statistical analyses were performed with the EZR software program (Saitama Medical Centre, Jichi Medical University). The data were assessed for significant Table 1 Nucleotide sequence for each primer
differences using the Bonferroni multiple comparison test, and a P value of \0.05 was considered to be statistically significant (*P \ 0.05, **P \ 0.01).
Gene
To confirm the potency of osteoclastogenesis induced by the supernatants of the stimulated PDL cells with TNF-a for 96 h, monocytes/macrophages were separately cultured in the differentiation medium containing the fivefold supernatants pretreated with the vehicle buffer (wild-type), control IgG (control), anti-IL-34 IgG (IL-34-deficient), anti-CSF-1 IgG2 (CSF-1-deficient) or anti-IL-34 and antiCSF-1 IgG2 (double deficient). A total of 7,535 ± 719 TRAP? cells and 132 ± 11 osteoclast-like cells, which contained more than three nuclei, were differentiated and matured from 20,000 monocytes/macrophages in the wildtype supernatants (Fig. 1a, b). The differentiation and maturation potencies of the wild-type supernatants were 40 % (J(7,535 TRAP? cells ? 132 osteoclast-like cells 9 3)/20,000 monocytes/macrophages 9 100) and 1.7 % (J132 osteoclast-like cells/(7,535 TRAP? cells ? 132 osteoclast-like cells 9 3) 9 100), respectively. The number of TRAP? cells and osteoclast-like cells changed to 7,355 ± 498 and 128 ± 10 following treatment with the control supernatants, 7,974 ± 1,093 and 49 ± 10 following treatment with the IL-34 deficient supernatants, 8,583 ± 798 and 48 ± 5 following treatment with the CSF-1-deficient supernatants and 8,163 ± 728 and 31 ± 5 following treatment with the double deficient supernatants, respectively. The differentiation and maturation potencies of the control supernatants and double deficient supernatants were 39 % (J(7,355 ? 128 9 3)/20,000 9 100) and 1.7 % (J128/(7,355 ? 128 9 3) 9 100) versus 41 % (J(8,163 ? 31 9 3)/20,000 9 100) and 0.4 % (J31/ (8,163 ? 31 9 3) 9 100), respectively. We assumed that the maturation potency of IL-34 and CSF-1 in the fivefold supernatants was 1.3 % (=1.7–0.4) in our experimental setting. Moreover, the differentiation and maturation potencies of the IL-34-deficient supernatants were 41 %
Primer sequence Forward
IL34 CSF1 GAPDH
Reverse
50 -GCCGACTTCAGTACATGAAACACT-30
50 -CCCTCGTAAGGCACACTGATC-30
0
0
50 -TTCGCGCAGTGTAGATGAAC-30
0
0
50 -ATGGTGGTGAAGACGCCAGT-30
5 -CATCCAGGCAGAGACTGACA-3 5 -GCACCGTCAAGGCTGAGAAC-3
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Fig. 1 Osteoclastogenesis potency of the PDL cell supernatants. Monocytes/macrophages were cultured for 12 days in the differentiation medium containing the fivefold supernatants of the stimulated PDL cells with TNF-a for 96 h pretreated with the vehicle buffer (minus, white bars), control rabbit IgG (plus, black bars), anti-IL-34 rabbit IgG (anti-IL-34, gray bars), anti-CSF-1 rabbit IgG (anti-CSF-1, hatched bars) or anti-IL-34 and anti-CSF-1 rabbit IgG (anti-IL-34/ anti-CSF-1, dotted bars) on 96-well culture plates (n = 7). a The
morphologically changed cells were stained with TRAP. b, c Five photographs were randomly taken to automatically count the number of TRAP? cells with a single nucleus as differentiated cells and the number of TRAP? cells with multiple nuclei as maturated (osteoclastlike) cells using the ImageJ program. The data were assessed for significant differences using the Bonferroni multiple comparison test, and a P value of \0.05 was considered to be statistically significant (*P \ 0.05, **P \ 0.01)
(J(7,974 ? 49 9 3)/20,000 9 100) and 0.6 % (J49/ (7,974 ? 49 9 3) 9 100), respectively, while the differentiation and maturation potencies of the CSF-1 deficient supernatants were 44 % (J(8,583 ? 48 9 3)/20,000 9 100) and 0.6 % (J48/(8,583 ? 48 9 3) 9 100), respectively. The maturation potencies of IL-34 (=1.3–0.6) and CSF-1 (=1.3–0.6) in the fivefold supernatants were the same at 0.7 %. These data indicate the synergistic effects of IL-34 and CSF-1 released from the stimulated PDL cells with TNF-a on the CSF-1R-mediated maturation of osteoclasts.
respectively. In contrast to the non-stimulated PDL cells, the stimulated PDL cells with TNF-a made the 52-fold IL34 (J259/5) and 20-fold CSF1 (J6,2561/3,122) transcripts. To confirm the autocrine mechanism underlying the gene expression of the CSF-1R ligands, we prepared a specific primer pair for the CSF1R gene. However, the amount of CSF1R transcripts was not changed by TNF-a (data not shown). To examine differences in the transcription sensitivities between promoter lesions of the IL34 and CSF1 genes, we collected total RNA from the stimulated PDL cells at 6, 24, 48 and 96 h after TNF-a loading. The relative gene expression levels of IL34 and CSF1 were 2.5 (J43 ± 4.3/ 17 ± 1.3) and 2.1 (J1.7 ± 0.1/0.8 ± 0.0) between 6 and 24 h, 1.2 (J52 ± 2.5/43 ± 4.3) and 11.8 (J20 ± 0.4/ 1.7 ± 0.1) between 24 and 48 h and 0.8 (J43 ± 1.0/ 52 ± 2.5) and 1.5 (J29 ± 0.0/20 ± 0.4) between 48 and 96 h, respectively (Fig. 2b). These data indicate that the IL34 and CSF1 genes in the PDL cells responded to the TNF-a-dependent production of the same transcription factors. However, the transcriptional sensitivity of the promoter lesions of the IL34 gene was higher than that of the CSF1 gene.
Gene expression levels of IL34 and CSF1 in the PDL cells To examine the gene expression levels of IL34 and CSF1 in our in vitro experimental setting, the transcripts in the stimulated PDL cells with the vehicle buffer or TNF-a for 48 h were measured using quantitative RT-PCR (Fig. 2a). The relative gene expression levels of IL34 and CSF1 to that of the control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with or without TNF-a were 5 ± 0.4 and 259 ± 51 as well as 3,122 ± 580 and 62,561 ± 1,553,
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Med Mol Morphol Fig. 2 IL34 and CSF1 gene expression in the PDL cells. a, b The IL34 and CSF1 transcripts in the PDL cells were measured at various time points after TNF-a loading using realtime PCR (n = 4). The results are expressed as the relative gene expression levels (2-DCt 9 104)
Protein production of IL-34 and CSF-1 in the PDL cells To confirm the level of protein production based on the above transcripts, the stimulated PDL cells with the vehicle buffer or TNF-a for 96 h were stained with or without antiIL-34 or anti-CSF-1 rabbit IgG. No control rabbit IgG deposition in the cytoplasm of the non-stimulated PDL cells was observed in our staining protocol (Fig. 3a left and b left). In the same staining protocol, anti-IL-34 rabbit IgG deposition was observed in the cytoplasm of the nonstimulated PDL cells and the stimulated PDL cells with TNF-a (Fig. 3a center and right). Meanwhile, anti-CSF-1 rabbit IgG deposition was also observed in the cytoplasm of the non-stimulated PDL cells and the stimulated PDL cells with TNF-a (Fig. 3b, center and right). To further confirm the extent of protein production in the stimulated PDL cells with TNF-a, apparent amounts of IL-34 and CSF-1 proteins relative to that of the control GAPDH proteins in the stimulated PDL cells for 96 h were measured using Western blotting. Consequently, the levels of IL-34 and CSF-1 proteins in the stimulated PDL cells with and without TNF-a were 0.8 ± 0.0 and 1.3 ± 0.0 versus 0.7 ± 0.0 and 1.1 ± 0.0, respectively (Fig. 3c). The PDL cells apparently produced approximately 1.6-fold
(J1.3/0.8) IL-34 and 1.6-fold (J1.1/0.7) CSF-1 proteins following treatment with TNF-a. Protein secretion of IL-34 and CSF-1 from the PDL cells To examine the level of protein secretion of IL-34 and CSF-1 from the PDL cells, the supernatants of the stimulated PDL cells with or without TNF-a for 96 h were collected, and the IL-34 and CSF-1 protein concentrations were measured using an enzyme-linked immunosorbent assay (Fig. 4). The relative amounts of IL-34 and CSF-1 proteins in the supernatants of the stimulated PDL cells with or without TNF-a were undetectable and 126.9 ± 83.8 pg/mL versus 177.0 ± 80.2 and 5,023.9 ± 1,073.9 pg/mL, respectively. The PDL cells apparently secreted approximately 28-fold CSF-1 proteins (J5,023.9/ 177.0) following treatment with TNF-a.
Discussion To assess the IL-34 production in the stimulated PDL cells with TNF-a, we evaluated the TNF-a-induced activation of
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Med Mol Morphol Fig. 3 IL-34 and CSF-1 protein expression in the PDL cells. a, b The stimulated PDL cells with the vehicle buffer for 48 h were stained with control rabbit IgG (white bars) (n = 6). The stimulated PDL cells with the vehicle buffer and TNF-a for 48 h were stained with anti-IL34 rabbit IgG (a, hatched bars and black bars) or anti-CSF-1 rabbit IgG (b, hatched bars and black bars), respectively (n = 6). c IL-34 and CSF-1 proteins in the stimulated PDL cells with the vehicle buffer and TNF-a for 48 h were detected using Western blotting with anti-IL-34 or anti-CSF-1 rabbit IgG (n = 6). The protein expression is expressed as the ratio of IL-34 and CSF-1 to GAPDH determined using the ImageJ 1.48 program
Fig. 4 IL-34 and CSF-1 protein secretion from the PDL cells. The levels of IL-34 and CSF-1 proteins in the supernatants of the stimulated PDL cells with the vehicle buffer or TNF-a for 96 h were measured using an enzyme-linked immunosorbent assay (n = 4)
transcription factors, such as NF-kB, AP-1, IRF-1 and NFGMa, and the transcriptional regulation of the IL34, CSF1 and CSF1R genes using the Searching Transcription Factor Binding Sites ver. 1.3 program (National Institute of Advanced Industrial Science And Technology, Tsukuba, Japan) [9]. The promoter sites of the IL34, CSF1 and CSF1R genes have the same binding motifs for transcription factors, GATA-1 and Myeloid Zinc Finger 1 (MZF1). However, we detected TNF-a-induced increments in the IL34 and CSF1 transcripts (Fig. 2a), but not the CSF1R transcripts (data not shown), in the stimulated PDL cells in our experimental setting. Therefore, AP-1 is the most suitable candidate transcription factor. The length from the binding position of AP-1 to ATG is shorter in the IL34
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gene than in the CSF1 gene. Therefore, our findings demonstrated the high sensitivity of the IL34 gene in the PDL cells relative to TNF-a in comparison with the CSF1 gene (Fig. 2b). CSF-1 appears to be localized in the cytoplasm of PDL cells, indicating the specific activation of the proteinreleasing pathway via the endoplasmic reticulum and Golgi complex (Fig. 3b) [18, 19]. Large numbers of localization motifs for secretory granules (EGRRs) or the extracellular space and Golgi apparatus (RRS, GSGH, FNETKN and so on) for CSF-1 (NP_757351.1) were searched using the Eukaryotic Motif resource for Functional Sites in Proteins (Bioinformatics Resource Portal), while a relatively small number of localization motifs for endoplasmic reticulum-
Med Mol Morphol Fig. 5 Maintenance potency of IL-34 or CSF-1. Monocytes/ macrophages were plated at 2 9 104 cells/well in 96-well culture plates and cultured for 12 days in the culture medium plus RANKL (20 ng/mL) containing with the vehicle buffer, recombinant IL-34 (100 ng/mL) or CSF-1 (100 ng/ mL) (n = 3)
Golgi transport vesicle membrane (YRSR) or the extracellular space and Golgi apparatus (KALLD) were found for IL-34 (NP_001166243.1). This information indicates that the affinity to the protein-releasing pathway of IL-34 is weaker than that for CSF-1 in PDL cells. Therefore, we observed the apparently ubiquitous expression of IL-34 in the cytoplasm of the stimulated PDL cells with TNF-a (Fig. 3a) [20]. We assumed the maturation potency of the fivefold supernatants to be 1.3 % for IL-34 and CSF-1 together and 0.7 % for each individually in our experimental setting (Fig. 1). Consequently, we found 126.9 ± 83.8 pg/mL of IL-34 and 5,023.9 ± 1,073.9 pg/mL of CSF-1 in the supernatants of the stimulated PDL cells with TNF-a for 96 h (Fig. 4). The dimer form of IL-34 commonly makes the CSF-1R dimer [2]. Therefore, 13 ng/mL of CSF-1 (J5,023.9 9 5/2) appears to bind CSF-1R on 147 TRAP? cells (=49 9 3) (Figs. 1, 4), while 0.3 ng/mL of IL-34 dimer (J126.9 9 5/2) appears to bind one set of unbound CSF-1R on 144 TRAP? cells (=48 9 3). In this study, we were unable to clarify the 80-fold high maturation potency of IL-34 in comparison to that of CSF-1. The effects of the bridging function of the IL-34 dimer on CSF-1R are not adequate to explain the differences in the maturation potency of the CSF-1R ligands. Conversely, the potency of the control supernatants (41 %) in stimulating differentiation to monocytes/macrophages was not changed by the IL-34-deficient supernatants, although it was significantly decreased to 44 % by the CSF-1-deficient supernatants (Fig. 1). Moreover, the duration until apoptotic death among the monocytes/macrophages in the differentiation medium treated without TNF-a was prolonged by recombinant IL-34, in contrast to the results observed by CSF-1 (Fig. 5). The IL-34 concentration in the supernatants of the non-stimulated PDL cells was under the limit of detection (Fig. 4). However,
our findings strongly suggested IL-34 production in the non-stimulated PDL cells, at least in our experimental setting. This hypothesis fits well with the concept that IL34 contributes to the survival of monocytes/macrophages during osteoclast differentiation [21]. PDL cells are well known to originate from the dental sac [22]. However, the prepared PDL cells appeared to be polyclonal. In this experiment, we did not address the characteristics of the prepared PDL cells. Conversely, periodontal ligament-specific markers were recently reported make it possible to distinguish PDL fibroblast-like cells from gingival fibroblasts [23]. We need to carry out further examinations to elucidate the specific roles of IL-34 released from PDL cell subpopulations in CSF-1R-mediated osteoclast maturation.
Conclusion Periodontal ligament cells secret low amounts of IL-34 and relatively high amounts of CSF-1 for the minimum maintenance of osteoclast precursor cells. When TNF-a is present, PDL cells might quickly secret IL-34 before CSF1 to initiate CSF-1R-mediated osteoclast differentiation and maturation. In this report, we demonstrated the coproduction of IL-34 and CSF-1 in stimulated PDL cells with TNF-a in our in vitro experimental setting. Acknowledgments This study was supported in part by a Grant-inAid for Researchers, Hyogo College of Medicine and Grant-in-Aid for Scientific Research (C) (No. 26463150 and No. 26463151) from the Japan Society for the Promotion of Science. We have no competing interests. The protocol and consent guidelines for the experiments were approved by the Human Ethics Committee of the Hyogo College of Medicine (Approval No. 223) according to the Declaration of Helsinki. All subjects provided their informed consent for the use of their tissues for research in this study.
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