Human Periodontal Ligament CellsSecrete Macrophage Colony-StimulatingFactor in Response to Tumor NecrosisFactor-Alpha In VitroTussanee Yongchaitrakul,* Kanokporn Lertsirirangson,* and Prasit Pavasant*

Background: Human periodontal ligament (HPDL) cellsmay support osteoclastogenesis by expressing receptor acti-vator of nuclear factor-kappa B ligand (RANKL) in responseto periopathogenic factors and inflammatory cytokines. Be-cause osteoclastogenesis requires the presence of macro-phage colony-stimulating factor (M-CSF), we examinedwhether HPDL cells secrete M-CSF in response to tumor ne-crosis factor-alpha (TNF-a).

Methods: Cultured HPDL cells were treated with TNF-a inserum-free condition. The expression of M-CSF and RANKLwas determined by reverse transcription-polymerase chain re-action and enzyme-linked immunosorbent assay. Inhibitorsand anti-TNF receptor (TNFR) neutralizing antibodies wereused for the inhibitory experiments. A migration assay wasperformed.

Results: TNF-a upregulated M-CSF and RANKL in HPDLcells. The effect on M-CSF expression could be partiallyblocked by pyrrolidine-dithiocarbamate ammonium salt andLY294002 but not by NS398. Neutralizing antibody toTNFR1 could diminish the effect of TNF-a. In addition, TNF-treated culture medium exhibited chemotactic effect forRAW264.7.

Conclusions: HPDL cells are capable of secreting M-CSFand expressing RANKL in response to TNF-a. The upregula-tion of M-CSF is possibly one of the mechanisms essentialfor periodontal tissue destruction in response to inflammatorycytokines. The upregulation is partly through nuclear factor-kappa B (NF-kB) and phosphatidylinositol 39-kinase and pos-sibly involves TNFR1. J Periodontol 2006;77:955-962.

KEY WORDS

Macrophage colony-stimulating factor; periodontal ligament;tumor necrosis factor-alpha.

Periodontitis is an inflammatory dis-ease that results in the destructionof the supporting connective and

bony tissues of the teeth. It has beenevidenced that human periodontal liga-ment (HPDL) cells are exposed to peri-opathogenic factors and inflammatorycytokines. Previous studies suggestedthat inflammatory cytokines and bacte-rial products could stimulate the expres-sion of proteinases from HPDL cells.1-3

In addition, HPDL cells respond to anactivation of interleukin-1 (IL-1), bacte-rial lipopolysaccharide, and compressiveforce by increasing receptor activator ofnuclear factor kappa B ligand (RANKL),which is involved in the activation anddifferentiation of osteoclasts.4-7 Thesefindings support a role of HPDL cells inthe process of tissue destruction andbone resorption.

Tumor necrosis factor-alpha (TNF-a),a potent osteoclastogenesis agent, hasbeen implicated in bone loss and con-nective tissue destruction associated withperiodontal disease.8-10 TNF-a is se-creted by inflammatory cells, gingival fi-broblasts, and PDL cells.9-12 It is thoughtto play a role in stimulating the productionof matrix metalloproteinases (MMPs).3

Because these MMPs are recognized toparticipate in tissue degradation andthe level of TNF-a is increased at the in-flammatory site, response of PDL cells tothe elevated levels of TNF-a may reflect

* Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Pathumwan,Bangkok, Thailand.

doi: 10.1902/jop.2006.050338

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an important mechanism in the process of alveolarbone destruction.

HPDL cells possibly play a role in osteoclastogen-esis through the expression of RANKL on their cell sur-faces.13-15 It is widely accepted in a model of bonecells that formation of osteoclasts requires the inter-action between receptor activator of nuclear factor-kappa B (NF-kB or RANK), which is expressed onthe surface of osteoclasts and their ligand, RANKL,in the presence of macrophage colony-stimulatingfactor (M-CSF).16

M-CSF is a growth factor that plays a role in theproliferation and differentiation of mononuclear phag-ocytes.17 This growth factor is also essential for osteo-clastogenesis because it facilitates the differentiationof osteoclast precursors.18,19 It is produced by bonemarrow stromal cells as soluble and membrane-associated factors and acts as a stimulating factorfor proliferation and differentiation of hematopoieticosteoclast precursors and promotes osteoclast for-mation and bone resorption.16 Induction of M-CSFby TNF-a has been reported in marrow stromal andgranulosa cells.20,21 Upregulation of M-CSF in HPDLcells stimulated by vitamin D3 was also reported byKimoto et al.22 However, the correlation betweenTNF-a and M-CSF in periodontal disease is still un-clear. We hypothesized that HPDL cells secretedM-CSF in response to TNF-a to facilitate the processof osteoclastogenesis. The purpose of this study wasto examine the effect of TNF-a on the production ofM-CSF in HPDL cells.

MATERIALS AND METHODS

Cell CultureHuman PDL cells were obtained from healthy thirdmolars extracted for orthodontic reasons and pre-pared as previously described.23 Informed consentwas obtained from each patient before the cell culturewas performed. Donors (one male and two females,aged 18 to 22 years) were enrolled in the study be-tween February and June 2005 at the dental hospital,Faculty of Dentistry, Chulalongkorn University. Theprotocol was approved by the ethical committee ofthe Faculty of Dentistry, Chulalongkorn University.Briefly, teeth were rinsed with sterile phosphate buff-ered saline (PBS) several times, and the PDL cellswere removed from the middle third of the root. Theexplants were harvested on a 60-mm culture dishand grown in Dulbecco’s modified Eagle’s medium†

supplemented with 10% fetal calf serum,‡ 2 mM L-glutamine,§ 100 units/ml penicillin,i 100 mg/mlstreptomycin,¶ and 5 mg/ml amphotericin B# at37�C in a humidified atmosphere of 95% air and 5%CO2. Cells from the third to the fifth passage wereused. All experiments were performed in triplicate us-ing cells prepared from three different donors.

Treatment of HPDL CellsHuman PDL cells were seeded in six-well plates at adensityof25,000cells/cm2andwereallowedtoattachfor 16 hours. To examine the effect of TNF-a on HPDLcells, 0.1, 1, and 10 ng/ml TNF-a** were added toeach well in serum-free medium for 24 to 48 hours.The medium and cell extracts were then collected foranalysis. The effective minimal dose of TNF-a was se-lected and used for the rest of the experiment.

In the inhibitory experiment, cells were treated withinhibitors for 30 minutes prior to addition of 1 ng/mlTNF-a. The inhibitors used in the experiment were1.4 mM LY294002,†† a phosphatidylinositol 39-kinase(PI3K) inhibitor, 50 mM pyrrolidine-dithiocarbamateammonium salt (PTDC),‡‡ an NF-kB inhibitor, and20 mM NS398, a cyclooxygenase-2 (COX-2) inhibi-tor. Cell extracts and the culture medium were thencollected after 24 hours of the treatment.

To determine whether TNF-a exerted its effect viaTNF receptors (TNFRs), namely, type 1 or p55r andtype 2 or p75r, the inhibitory antibodies to TNFR1§§

(mouse monoclonal immunoglobulin G1 againsthuman TNFR1) and TNFR2ii (mouse monoclonal im-munoglobulin G2A against human TNFR2) were used.Cells were pretreated with 6 mg/ml anti-TNFR1 anti-body or 2 mg/ml anti-TNFR2 antibody before beingtreated with 1 ng/ml TNF-a for another 24 hours.

RNA Extraction, cDNA Synthesis, andSemiquantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)After the treatment, total cellular RNA was extractedwith reagent¶¶ according to the manufacturer’s in-structions. The concentration of purified RNA in eachsample was determined by the absorption at 260/280 nm. One microgram of each RNA sample wasconverted to cDNA by reverse transcription using anavian myeloblastosis virus## reverse transcriptase for1.5 hours at 42�C. Subsequent to the RT, PCR wasperformed. The primers were prepared following thereported sequences from GenBank. The oligonucleo-tide sequences of the primers are shown in Table 1.

The PCR was performed using polymerase*** witha PCR volume of 25 ml. The mixtures contained 25pmol primers and 1 ml RT product. The PCR workingconditions were set at a denaturation for 1 minute

† Gibco BRL, Carlsbad, CA.‡ Gibco BRL.§ Gibco BRL.i Gibco BRL.¶ Gibco BRL.# Gibco BRL.** Sigma-Aldrich Chemical, St. Louis, MO.†† Cayman Chemical, Ann Arbor, MI.‡‡ R&D Systems, Minneapolis, MN.§§ R&D Systems.ii R&D Systems.¶¶ TRI reagent, Molecular Research Center, Cincinnati, OH.## Promega, Madison, WI.*** Tag polymerase, Qiagen, Hilden, Germany.

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at 94�C, primer annealing for 1 minute at 60�C, andchain elongation for 1.45 minutes at 72�C on a DNAthermal cycler.††† The amplified DNA was thenelectrophoresed on a 2% agarose gel and visualizedby ethidium bromide fluorostaining. The intensityof each band was determined by image analysissoftware.‡‡‡

Western Blot AnalysisCells were treated with various doses of TNF-a for 48hours and extracted using RIPA buffer (150 mM NaCl;1% NP-40; 0.5% deoxycholate; 0.1% sodium dodecylsulfate [SDS]; 50 mM Tris, pH 8.0) containing cocktailprotease inhibitors.§§§ The amount of proteins wasdetermined using a protein assay kit.iii Proteinextraction from each sample was subjected toSDS-polyacrylamide gel electrophoresis under a re-ducing condition on a 12% polyacrylamide gel. Afterelectrophoresis, proteins were electrophoreticallytransferred onto a nitrocellulose membrane.¶¶¶ Themembrane was incubated in blocking buffer (5%non-fat dry milk and 0.1% Tween 20 in deionizedwater) at room temperature for 1 hour. The mem-brane was stained overnight with primary antibodyfor RANKL### or b-actin**** at a dilution of 1:500in blocking buffer at 4�C. After extensive washing withPBS, the membrane was incubated with biotinylated-secondary antibody†††† for 30 minutes at room tem-perature and peroxidase-conjugated streptavidin‡‡‡‡

for 30 minutes. The protein bands were detected usinga chemiluminescence system§§§§ and exposed onfilm.iiii The band intensity was determined by imageanalysis software.¶¶¶¶

Enzyme-Linked Immunosorbent Assay (ELISA)The amount of M-CSF in the culture medium wasmeasured using an ELISA kit#### following the manu-facturer’s instructions. Triplicate assays per experi-ment were performed.

Chemotaxis AssayHPDLcells were cultured withorwithout1 ng/mlTNF-ain serum-free medium. After 24 hours, the culturemedium was then changed to serum-free conditionwithout TNF-a to eliminate the effect of TNF-a itself.Cells were incubated for another 24 hours, followed bya collection of the conditioned medium. The chemo-tactic effect of the conditioned medium on osteoclastprecursor cell line RAW264.7 was assayed using a48-well chemotaxis chamber. RAW264.7 cells (56,000cells/56 ml) were seeded onto the upper chamberand allowed to migrate across the membrane for 20hours. Cells were fixed with 4% paraformaldehydefor 30 minutes, followed by a brief wash in PBS, andstained with hematoxylin for 10 minutes. The mem-brane was placed on the glass slide, and the cells werecounted under the microscope.

Statistical AnalysisData are expressed as mean – SD. Statistical signifi-cance was determined by analysis of variance, andScheffe’s test was used for post-hoc analysis. A Pvalue <0.05 was considered statistically significant.

RESULTS

The effect of TNF-a (0.1, 1, and 10 ng/ml) on the ex-pression of M-CSF and RANKL in HPDL cells wasexamined. RT-PCR analysis revealed the increasedexpression of M-CSF mRNA in a dose-dependentmanner as shown in Figure 1A. The amount ofM-CSF in the culture medium at 24 hours after thetreatment was also determined by ELISA. The datashowed a significant increase (P <0.05) of M-CSF inthe culture treated with 1 and 10 ng/ml TNF-a (Fig.1B). In addition, the expression of RANKL mRNA alsoincreased in a dose-dependent manner (Fig. 2A). Thealteration of protein was confirmed by Western blotanalysis (Fig. 2B). The results represent one of thetriplicate experiments. One nanogram/milliliter ofTNF-a was consequently selected and used for therest of the study.

To examine the involvement of COX-2 in the upreg-ulation of M-CSF and RANKL in HPDL cells by TNF-a,cells were treated with NS398, a COX-2 inhibitor,30 minutes prior to the treatment. RT-PCR analysisrevealed that NS398 could inhibit the effect ofTNF-a on RANKL mRNA expression. By contrast,

Table 1.

Sequences of Forward and ReversePrimers of Target Genes

Target

mRNA Primer Sequence (59 to 39)

M-CSF Forward: CTA AGC TGG ACG CAC AGA CCA

Reverse: TCT CAG GCT GCA CAC CTT

RANKL Forward: CCA GCA TCA AAA TCC CAA GT

Reverse: CCC CTT CAG ATG ATC CTT C

GAPDH Forward: TGA AGG TCG GAG TCA ACG GAT

Reverse: TCA CAC CCA TGA CGA CGA ACA TGG

††† ThermoHybaid, Ashford, U.K.‡‡‡ Scion, Frederick, MD.§§§ Sigma-Aldrich Chemical.iii BCA kit, Pierce, Rockford, IL.¶¶¶ Millipore, Bedford, MA.### Chemicon International, Temecula, CA.**** Chemicon International.†††† Sigma-Aldrich Chemical.‡‡‡‡ Chemicon International.§§§§ Millipore.iiii Millipore.¶¶¶¶ Scion.#### R&D Systems.

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the enhancement effect on M-CSF mRNA expressioncould not be blocked by NS398, indicating a distinctsignaling pathway. The intensity of each band nor-malized to an equal amount of GAPDH mRNA is rep-resented as a graph shown in Figure 3.

Upregulation of RANKL mRNA and protein in HPDLcells via the COX-dependent pathway has been previ-ously reported.7 Our results in regards to increasedRANKL production by TNF-a are similar to those ofothers.4,7,22 Therefore, we analyzed the pathway ofM-CSF stimulation by TNF-a. We further investigatedthe intracellular signaling pathway that may be in-volved in the regulation of M-CSF using inhibitoryreagents. The expression and secretion of M-CSFinduced by TNF-a appeared to be diminished byLY294002 and by PTDC as shown in Figure 4.

To assess which TNFRs of HPDL cells are involvedin responding to TNF-a, neutralizing antibody toTNFR1 and TNFR2 was added. The result demon-strated that TNFR1, but not TNFR2, partially blockedthe effect of TNF-a, suggesting the involvement ofTNFR1 in the enhancement of M-CSF. Figure 5 repre-sents one of triplicate experiments.

Because M-CSF has been reported to induce mi-gration of macrophages, we hypothesized that theconditioned medium collected from TNF-treated cul-ture that contained HPDL cell–secreted molecules, in-

cluding M-CSF, might exhibit chemotactic effect.Thus, we further tested the effect of the conditionedmedium with RAW264.7 using a chemotaxis cham-ber. As shown in Figure 6, the TNF-treated culturemedium from HPDL cells induced the migration ofRAW264.7 cells up to five-fold compared to the con-trol. This result indicated that the secreted moleculesinduced by TNF-a could promote the migration ofRAW264.7.

DISCUSSION

TNF-a is one of the molecules implicated in peri-odontal disease. It has been known to stimulate highproduction of RANKL leading to osteoclastogenesisand bone destruction. The present data revealed thatHPDL cells responded to TNF-a by upregulating notonly the expression of RANKL but also M-CSF, thefactor that plays a role in the survival, proliferation,and differentiation of monocyte-macrophage lineage,including osteoclasts.17

The mechanism ofTNF-a on the induction ofM-CSFwas also investigated. Our study suggests that the in-ductionofM-CSFbyTNF-awassignaledpartly throughTNFR1. This result corresponded to the results from

Figure 2.Analysis of RANKL by RT-PCR and Western blot. HPDL cells weretreated with a series of doses of TNF-a (0.1, 1, and 10 ng/ml) for24 hours. A)The PCR products reveal an increase of RANKLexpression observed at 1 and 10 ng/ml. B) An alteration of theprotein of RANKL in corresponding PCR products. Cells werecultured in serum-free medium for 48 hours. Cell extractswere collected and confirmed by Western blot analysis. Actinwas used as an internal control.

Figure 1.An analysis of M-CSF by RT-PCR and ELISA. A) The expression ofM-CSF in HPDL cells after treatment with 0.1, 1, and 10 ng/mlTNF-a in serum-free medium for 24 hours. The expression of M-CSFincreased in a dose-dependent manner. GAPDH is used as internalcontrol. B) Relative level of M-CSF assayed by ELISA. *Statisticaldifference (P <0.05). The figure represents one of three experiments.

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others showing that TNFR1 is the major receptor forTNF-a.24,25 A review by Chen and Goeddel26 indicatedthat the signal from TNFR1 could activate a cascade ofkinases, resulting in the activation of NF-kB and c-Jun.We have also investigated these pathways on the TNF-inducedM-CSFexpression.Wefoundthatthe inductionof M-CSF partly involved PI3K and NF-kB because theexpression was partially blocked by LY294002 andPTDC. However, unlike the upregulation of RANKL,which has been shown to occur through a COX-dependent pathway, the present results indicated thatCOX-2 did not participate in the induction of M-CSFby TNF-a in HPDL cells.

M-CSF is the most pleiotropic macrophage growthfactor. The function of this growth factor is not only in-volved in the proliferation and differentiation of cellsin the monocyte-macrophage lineage but also facili-tates the spreading and motility of macrophage.27-29

Expression of this growth factor can be modified byseveral inflammatory cytokines. TNF-a and IL-4 arepotent stimulators of M-CSF synthesis by bone mar-row stromal cells.20 The work by Kawano et al.21 usinghuman granulosa cells revealed that both IL-1 andTNF-a could increase the secretion of M-CSF. Inaddition, Tanabe et al.30 showed that IL-1 could stim-ulate M-CSF production in osteoblast-like cells, ROS17-2.8. In this study, we provided evidence that TNF-aincreased the secretion and mRNA expression ofM-CSF in HPDL cells.

The upregulation of M-CSF by TNF-a

in HPDL cells might support the differen-tiation and the survival of osteoclasts.M-CSF has been shown to be an essen-tial factor in addition to RANKL for thedifferentiation of osteoclasts. The worksreported by Suda et al.16 and Tanakaet al.17 showed that the signal from M-CSF is one of the crucial factors requiredfor the differentiation of osteoclast pro-genitors. Furthermore, M-CSF alsoinvolves the transdifferentiation of den-dritic cells into osteoclasts.31 The workof Rivollier et al.31 suggested that osteo-clasts derived from dendritic cells mayplay an important role in the pathogenicbone resorption, and M-CSF is one of therequirements for the transdifferentia-tion. In osteopetrotic mice, which havea mutation in the coding region of theM-CSFgene,aseveredeficiencyofoste-oclasts that results in the congenital os-teopetrosis was observed.32,33 M-CSFcan also promote the survival of osteo-clasts. Glantschnig et al.34 reported thatsignaling by M-CSF, and the signal fromRANKL and TNF-a, was important for

Figure 3.PCR products show the effect of NS398 on the expression ofM-CSF and RANKL in HPDL cells. NS398 could block the expressionof RANKL but not that of M-CSF in the cultures stimulated with1 ng/ml TNF-a for 24 hours. The density of each band wasdetermined by image analysis software, and the comparativeintensity normalized to an equal density of GAPDH is representedas a graph.

Figure 4.Analysis of M-CSF in the inhibitory experiment by RT-PCR and ELISA. A) Expression ofM-CSF in HPDL cells after treatment with 1 ng/ml TNF-a, 50 mM PI3K inhibitor(LY294002), 50 mM PTDC, or the combination of TNF-a and either of the inhibitors for24 hours. The relative intensity of each band normalized to equal density of GAPDH,determined by image analysis software, is shown as a graph. The graph in B shows therelative amount of M-CSF in the media measured by ELISA. The figure represents oneof the triplicate experiments.

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the survival of osteoclasts because it showed anti-apoptotic activity through mTOR/S6 kinase in osteo-clasts.

M-CSF also plays a role in macrophage motilityand acts as a chemoattractant for macrophage.28 Re-

garding osteoclasts, Fuller et al.27

showed that M-CSF is involved in themotility of osteoclast isolated fromneonatal rat long bones. In this study, wealso found that secreted molecules fromHPDL cells treated with TNF-a could in-duce the migration of RAW264.7 cellsacross the membrane in the migrationassay. We hypothesized that increasinglevels of M-CSF in the culture mediummight be involved in this chemotacticeffect. However, we have no direct prooffor our hypothesis at this time, and thisissue needs further investigation. How-ever, we would like to suggest that theinduction of M-CSF by TNF-a mightbe involved in the recruitment of phago-cytic cells, including osteoclast precur-sors, into the periodontal space. Thisrecruitment might be one of the earlyphenomena of the pathogenesis of per-iodontal disease leading to alveolarbone destruction.

HPDL cells are known to participatein periodontal regeneration and de-struction. Biologically, these cells func-tion as host cells that try to keep a

balance between regeneration and degradation. Aprevious study reported that HPDL cells were capableof secreting osteoprotegerin, which opposed the func-tion of RANKL.13 We found that cells from fresh per-iodontal ligament collected from the freshlyextracted teeth also expressed M-CSF (data notshown). Existence of M-CSF in a normal conditioncould be a function of keeping a balance among themolecules that augment or oppose osteoclast forma-tion. However, a significant increase of M-CSF byHPDL cells in response to TNF-a suggests that thecells secrete M-CSF as another paracrine factor thatfacilitates bone destruction or remodeling.

Our results indicated that COX-2 was one of the sig-naling molecules participating in the upregulation ofRANKL by TNF-a. It is interesting to note that severalreports demonstrated the COX-2/prostaglandin E2

(PGE2) pathway in the regulation of RANKL in manycell types, including HPDL cells.17,35-37 Hence, thesignal from COX-2/PGE2 products seems to be amajor pathway involving RANKL expression.

CONCLUSIONS

Our study reveals that TNF-a can stimulate the ex-pression and secretion of M-CSF from HPDL cells invitro. The response of HPDL cells to TNF-a by increas-ing M-CSF in addition to RANKL emphasizes the roleof HPDL cells in the pathogenesis of periodontal dis-ease. The upregulation of M-CSF may participate

Figure 5.Detection of M-CSF of the cultures treated with TNF-a and inhibitory antibodies toTNFR1 or TNFR2. RT-PCR analysis reveals an inhibitory effect of the antibody to TNFR1but not to TNFR2. The relative density is presented as a graph (A). The graph in B showsthe amount of M-CSF in the media measured by ELISA. The results represent one of thetriplicate experiments.

Figure 6.The graph demonstrates the number of RAW264.7 cell linesmigrated across the membrane in the chemotactic assay using achemotaxis chamber. HPDL cells were cultured with or without1 ng/ml TNF-a in serum-free medium for 24 hours, and the mediumwas collected to test for chemotactic effect. TNF-treated conditionedmedium (CM) enhanced cell migration up to five-fold compared tothe control.

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in the recruitment and differentiation of osteoclastprecursors and survival of osteoclasts, thereby facili-tating periodontal tissue destruction.

ACKNOWLEDGMENTS

The authors thank Dr. Kim Mansky, University ofMinnesota, St. Paul, Minnesota, for providing theRAW264.7 cell line. This work was supported by theRatchadaphisek Somphot Endownment, Chulalong-korn University, Bangkok, Thailand.

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Correspondence: Dr. Prasit Pavasant, Department of Anat-omy, Faculty of Dentistry, Chulalongkorn University,Henri-Dunant Rd., Pathumwan, Bangkok 10330, Thailand.Fax: 66-2-218-8870; e-mail: prasit_215@yahoo.com.

Accepted for publication December 7, 2005.

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