Australian Dental Journal
The official journal of the Australian Dental Association
Australian Dental Journal 2015; 60: 382–389 doi: 10.1111/adj.12234
Upregulated expression of monocyte chemoattractant protein-1 in human periodontal ligament cells induced by interleukin-1b J Jin,* J Cao* *State Key Laboratory of Military Stomatology, Department of Orthodontics, School of Stomatology, the Fourth Military Medical University, Xi’an, China.
ABSTRACT Background: Root resorption during orthodontic treatment is a complex and sterile inflammatory process, characterized by the recruitment of mononuclear cells in the local periodontal ligament. This study aimed to investigate whether interleukin (IL)-1b could induce the migration of monocytes through upregulating monocyte chemoattractant protein (MCP)1 expression in human periodontal ligament cells. Methods: Human periodontal ligament cells were cultured in medium containing various IL-1b concentrations. After 24 hours of incubation, the messenger RNA (mRNA) and MCP-1 protein in periodontal ligament cells were detected by reverse transcriptase–polymerase chain reaction and Western blot analysis, respectively. The effect of supernatants of periodontal ligament cells on THP-1 cells was analysed via migration assay. Results: IL-1b (10 ng/mL and 25 ng/mL) increased the expression of MCP-1 mRNA and protein in periodontal ligament cells (p < 0.05). In addition, the supernatants from the IL-1b-treated periodontal ligament cells increased the migratory response of THP-1 cells (p < 0.05), which could be blocked by the anti-MCP-1 antibody. Conclusions: IL-1b had the potential to induce the migratory response of monocytes via upregulation of the expression of MCP-1 in human periodontal ligament cells and could contribute to orthodontic root resorption. Keywords: Chemotaxis, interleukin-1b, monocyte, monocyte chemoattractant protein-1, periodontal ligament cell. Abbreviations and acronyms: DMEM = Dulbecco’s Modified Eagle’s Medium; FBS = fetal bovine serum; IL = interleukin; MCP = monocyte chemoattractant protein; M-CSF = macrophage colony-stimulating factor; PCR = polymerase chain reaction; PDL = periodontal ligament; RANKL = receptor activator of nuclear factor kappa-B; RIPA = radioimmunoprecipitation assay; RT-PCR = reverse transcription–polymerase chain reaction. (Accepted for publication 2 October 2014.)
INTRODUCTION Root resorption, the unwanted side effect following orthodontic tooth movement, is triggered by a sterile inflammatory process.1–3 The inflammatory process is characterized by mononuclear cell recruitment. Chemokines and proinflammatory cytokines released by periodontal ligament (PDL) cells underlie the inflammatory state. Interleukin (IL)-1b is a proinflammatory cytokine and a potent stimulus for bone resorption and osteoclastic cell recruitment during orthodontic tooth movement.4 Diseases that affect the properties of bone often alter the metabolism of cementum since they have a similar composition.5 Increased levels of IL-1b have been found at the compression side of the PDL during orthodontic tooth movement.3,6,7 A combina382
tion of loading and an increased level of IL-1b has been discussed as being responsible for the loss of cementum.6 Human primary cementoblasts subjected to compression and IL-1 stimulation could impede some cementogenesis-associated proteins expression, such as bone sialoprotein and cementum-derived protein.7 A role for IL-1b in the pathogenesis of root resorption is suggested since the polymorphism of the IL-1b gene is associated with root resorption.4,8 During the root resorption process, the PDL cells are constantly stimulated by increasing IL-1b. Chemokines direct leukocyte trafficking and positioning within the tissues, and they play critical roles in regulating immune responses and inflammation. Chemokines are a large family of small proteins, which play the crucial role of chemotaxis in cell trafficking, angiogenesis, cell proliferation, apoptosis, tumour metastasis © 2015 Australian Dental Association
MCP-1 expression in PDL cells induced by IL-1b and host defense.9,10 As a potent chemokine for monocytes, monocyte chemoattractant protein (MCP)-1 is implicated in the pathogenesis of diseases characterized by monocyte infiltration.10–14 It was demonstrated that MCP-1 presented in the PDL cells in the root resorption area,15 which implied that monocytes were probably directed to migrate via signals from MCP-1, which was released from resident cells of PDL during the resorption process. It has been shown that IL-1b can induce MCP-1 expression in many resident cells, such as chondrocytes and osteoblastic cells.16,17 However, there is no direct evidence for the role of IL-1b in MCP-1 expression in PDL cells. A singular differentiation process within the haematopoietic system is represented by the differentiation of monocytes to osteoclasts. Osteoclasts are giant, multinucleated cells, and are specialized in degrading bone.18 Since cementum and bone have a similar composition, mononuclear cells are thought to play a role in root resorption.5 When stimulated with receptor activator of nuclear factor kappa-B (RANKL) and macrophage colony-stimulating factor (M-CSF), mononuclear cells could differentiate into macrophages or fuse to form odontoclasts.19–25 Additionally, factors produced by mononuclear cells may contribute to resorption or injury.26,27 Despite the potential importance of mononuclear cells in root resorption, the molecular mechanism involved in the recruitment of monocytes has been unclear, until now. The present study aimed to investigate the expression of MCP-1 in PDL cells induced by IL-1b, as well as the migration of THP-1 cells regulated by the expression of MCP-1. It was shown that IL-1b could increase the migratory response of monocytes via upregulation of the expression of MCP-1. This may be one of the mechanisms through which monocyte recruitment occurs during orthodontic root resorption. METHODS Cell culture PDL cells were obtained from 20 teeth of five patients (12–13 years of age) following premolar extraction, as indicated during orthodontic treatment. The research protocol was approved by the Human Experimentation Committee of the School of Stomatology, the Fourth Military Medical University, People’s Republic of China. Informed consent was obtained from the patients’ parents after being provided with an explanation of the study. PDL cells were cultured as described previously.28 Briefly, the PDL was gently scraped off from the middle third of the root surface to avoid contamination from the gingival and apical © 2015 Australian Dental Association
tissues. The tissue explants were transferred to cell culture flasks containing Dulbecco’s Modified Eagle’s Medium (DMEM; Gibcoâ BRL, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with antibiotics (penicillin 100 U/mL, streptomycin 100 lg/ mL) and 10% fetal bovine serum (FBS; Gibcoâ BRL, Thermo Fisher Scientific, Waltham, MA, USA). The flasks were placed in a water-jacketed cell/tissue incubator with 5% CO2. The cells were allowed to migrate from the explants. After reaching confluence, the cells were detached by incubation with 0.25% trypsin and reseeded at a density of 80 000 cells/mL. PDL cells were used between passages three and five for the experiments. Cell treatment First, all the PDL cells from the five patients were mixed. The mixed PDL cells were then seeded in six-well plates at 1 9 106 cells/well. After reaching 80% confluence, the cells were serum starved with serum-free DMEM overnight to achieve standardized conditions. In the experimental group, the PDL cells were treated with various concentrations (1 ng/mL, 5 ng/mL, 10 ng/mL or 25 ng/mL) of recombinant human IL-1b (Sigma-Aldrich Company, St. Louis, MO, USA) in 3 mL of DMEM supplemented with 1% FBS for 24 hours. In the control group, the PDL cells were incubated in 3 mL of DMEM supplemented with 1% FBS for 24 hours. The cells in different treatment groups were then collected and used for reverse transcription–polymerase chain reaction (RT-PCR) analysis and Western blot analysis. The cell-free supernatants were stored at 70 °C for the migration assay. Reverse transcription–polymerase chain reaction analysis Total RNA from the PDL cells was extracted using TRIzolâ reagent (Gibcoâ BRL, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The RNA samples were reversetranscribed into first-strand complementary DNA (cDNA) using the SuperScriptâ Preamplification System (Gibcoâ BRL, Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was amplified by polymerase chain reaction (PCR). PCR was carried out in 50 lL of PCR reaction buffer containing 2.5 lL of the template, 1.0 U of Taq polymerase (Promega Corporation, Fitchburg, WI, USA), 0.2 mM of deoxyribonucleotide triphosphates (dNTPs), and 100 pmol forward and reverse primers. The reaction mixture was incubated at 94 °C for 50 seconds. It was then subjected to 30 cycles of denaturing at 94 °C for 35 seconds, annealed at 58 °C for 60 seconds, and extended at 72 °C for 383
J Jin and J Cao 30 seconds. The reaction was performed in duplicate. The sequences of PCR primers for MCP-1 were 50 -CT TCTGTGCCTGCTGCTCATA-30 (forward) and 50 -CT TTGGGACACTTGCTGCTG-30 (reverse). Those for GAPDH, a housekeeping gene, were 50 -CGGAGTCA ACGGATTTGGTCGTAT-30 (forward) and 50 -AGCC TTCTCCATGGTGGTGAAGAC-30 (reverse). The PCR products were resolved on 1.8% agarose gel and visualized by ethidium bromide staining. The relative intensities of the PCR products were analysed with picture imaging software (Scion Image, Informer Technologies, Inc., Frederick, MD, USA). The results were normalized to the corresponding GAPDH messenger RNA (mRNA) levels. Western blot analysis PDL cells in each well were washed with PBS, lysed with ice cold radioimmunoprecipitation assay (RIPA) buffer (Bio-Rad Laboratories Inc., Hercules, CA, USA), and centrifuged at 10 000 g for 5 minutes at 4 °C. The supernatants were collected and subjected to Western blot analysis. The protein content of the samples was measured using the BCA protein assay kit according to the manufacturer’s protocol (Pierce Chemical Company; Thermo Fisher Scientific, Waltham, MA, USA). Each protein sample (20 lg) was subjected to sodium dodecylsulfate–polyacrylamide electrophoresis (SDS-PAGE) and transferred from SDS gels onto a poly-vinyliden difluoride (PDVF) membrane for immunoblot analysis. The membranes were blocked with 5% BSA in Tris buffered saline with Tweenâ (TBST). The membrane was then incubated at 4 °C for 8 hours with rabbit anti-human MCP-1 antibodies (Abcam plc, London, UK) diluted 1:500 in 5% non-fat dry milk in TBST. After washing, the membranes were incubated at room temperature for 1 hour with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (IgG) (GE, Arlington Heights, IL, USA) diluted 1:3000 in 5% non-fat dry milk in TBST, and developed using the ECL system (GE, Arlington Heights, IL, USA). b-actin-specific antibody (1:1000; Abcam, London, UK) was used for loading controls on the stripped membranes. The relative protein expressions were analysed with picture imaging software (Scion Image; Informer Technologies, Inc., Frederick, MD, USA). The results were normalized to the corresponding b-actin expression levels. Migration assay The THP-1 cell line, derived from human acute monocytic leukaemia, was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI 1640 (Gibcoâ BRL; 384
Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% FBS, 100 U/mL of penicillin, 100 lg/mL of streptomycin and 2 mM of L-glutamine. Cell migration was assessed in 24-well chemotaxis chambers fitted with 8.0 lm transwell membranes (Corning, Rosemont, VA, USA). Then, 1 9 106 THP-1 cells in 100 lL RPMI 1640 containing 1% FBS were added to the upper wells. In the experimental groups, the supernatants (150 lL) of PDL cells cultured in medium with various IL-1b concentrations (1 ng/mL, 5 ng/mL, 10 ng/mL and 25 ng/mL) were added to the lower chamber of the transwell system. In the control group, the supernatant (150 lL) of PDL cells cultured in medium without IL-1b (0 ng/mL) was added. In the negative control group, the medium containing IL-1b was used instead of the supernatant. The medium was DMEM supplemented with 1% FBS. In the blocking experiments, the assay of THP-1 cell migration was performed using supernatants of PDL cells with or without 10 ng/mL of IL-1b treatment. In the blocking groups, the supernatants of PDL cells were incubated with 1 lg/mL anti-MCP-1 antibody (Abcam, London, UK) overnight at 4 °C
Table 1. Effect of various IL-1b concentrations on relative mRNA expression (MCP-1/GAPDH) in PDL cells (n = 4) IL-1b concentration (ng/mL)
0 1 5 10 25
Mean
0.80 0.80 0.80 0.95* 0.94*
SD
0.03 0.05 0.02 0.03 0.04
95% confidence interval Lower boundary
Upper boundary
0.77 0.75 0.78 0.92 0.91
0.83 0.85 0.82 0.98 0.97
*p < 0.05, compared with the control group (0 ng/mL IL-1b treatment).
Table 2. Effect of various IL-1b concentrations on relative protein expression (MCP-1/b-actin) in PDL cells (n = 4) IL-1b concentration (ng/mL)
0 1 5 10 25
Mean
0.41 0.41 0.42 0.92* 0.91*
SD
0.04 0.02 0.05 0.03 0.04
95% confidence interval Lower boundary
Upper boundary
0.37 0.38 0.38 0.89 0.87
0.44 0.43 0.47 0.96 0.95
*p < 0.05, compared with the control group (0 ng/mL IL-1b treatment). © 2015 Australian Dental Association
MCP-1 expression in PDL cells induced by IL-1b (b) IL-1β (ng/ml) 0
1
5
10
25
500 bp 250 bp
MCP-1 (166 bp)
100 bp 500 bp
GAPDH (306 bp)
250 bp 100 bp
1.2
Relative mRNA expression (MCP-1/GAPDH)
(a)
*
*
1 0.8 0.6 0.4 0.2 0
0 ng/ml 1 ng/ml 5 ng/ml 10 ng/ml 25 ng/ml
The concentration of IL-1β
Fig. 1 Effects of IL-1b on the MCP-1 mRNA expression in PDL cells. (a) PDL cells were exposed to various IL-1b concentrations (1–25 ng/mL), and total RNA isolated from the cells treated at each concentration was subjected to RT-PCR analysis. (b) The expression of each molecule shown in (a) was quantified by image analysis. The results are presented as the ratio of the mRNA level to GAPDH in each molecule. The means SD from four repeated experiments are reported. By ANOVA with Bonferroni’s post hoc test, significant differences compared to the control group (0 ng/mL; IL-1b treatment) are shown (*p < 0.05).
(a)
(b)
0
1
5
10
25
MCP-1
β-actin
Relative protein expression (MCP-1/β-actin)
IL-1β (ng/ml)
1.2 *
1
*
0.8 0.6 0.4 0.2 0 0 ng/ml 1 ng/ml 5 ng/ml 10 ng/ml 25 ng/ml The concentration of IL-1β
Fig. 2 Effects of IL-1b on the MCP-1 protein expression in PDL cells. (a) PDL cells were exposed to various IL-1b concentrations (1–25 ng/mL), and the whole-cell lysates were extracted, resolved on 8% SDS-PAGE, and subjected to Western blot analysis with an antibody against MCP-1. (b) Data in the graph were shown as the means SD from four repeated experiments. By ANOVA with Bonferroni’s post hoc test, significant differences compared to the control group (0 ng/mL; IL-1b treatment) are shown (*p < 0.05).
before the migration assay. In the control groups, the supernatants were without the anti-MCP-1 treatment. The transwell membranes were removed after 90 minutes of incubation at 37 °C. Non-migrated cells were then removed from the system. The migrated cells were fixed in 4% formaldehyde for 15 minutes at 37 °C and stained with crystal violet. The cell number in five random fields (original magnification: 4009) was counted. Statistics The RT-PCR and Western blot experiments were repeated four times, and the migration assay was performed nine times. The data of the results in the repeated experiments followed a normal distribution. The means, standard deviations (SD) and 95% confidence intervals were calculated. Statistical significance was calculated using analysis of variance (ANOVA) and Student’s two-tailed t-test for unpaired comparisons with the Bonferroni test for post hoc analysis, as appropriate. P-values less than 0.05 were regarded as statistically significant. © 2015 Australian Dental Association
RESULTS Effect of IL-1b on MCP-1 expression in PDL cells The means, standard deviations (SD) and 95% confidence intervals of MCP-1 mRNA and protein expression under various IL-1b concentrations are
Table 3. Effect of PDL cells stimulated with various IL-1b concentrations on THP-1 cell migration (n = 9) IL-1b concentration (ng/mL) Mean
0 1 5 10 25
31.11 62.22* 193.22* 342.22* 352.66*
SD
5.30 7.27 6.62 5.64 7.58
95% confidence interval Lower boundary
Upper boundary
25.81 54.95 186.60 336.58 345.08
36.41 69.49 199.84 347.86 360.25
*p < 0.05, compared with the control group (0 ng/mL IL-1b treatment). 385
J Jin and J Cao described in Tables 1 and 2. As shown in Fig. 1, PDL cells expressed MCP-1 mRNA at basal levels without IL-1b stimulation. Treatment with IL-1b could increase the MCP-1 mRNA expression of PDL cells, with no effect at 1 ng/mL and 5 ng/mL (p > 0.05), and significant effects were observed at 10 ng/mL and 25 ng/mL (p < 0.05). Similar results were found for the MCP-1 protein by Western blot analysis (Fig. 2). Though the expression of MCP-1 protein in the PDL cells without IL-1b stimulation could be detected at low levels, IL-1b with higher concentrations (10 ng/mL and 25 ng/mL) increased the protein expression significantly (p < 0.05). IL-1b at lower concentrations (1 ng/mL and 5 ng/mL) could not significantly change the MCP-1 expression in PDL cells (p > 0.05). These results demonstrated a bioactive role of IL-1b in modulating the MCP-1 expression of PDL cells.
Table 4. The blocking effect of anti-MCP-1 on THP-1 cell migration induced by supernatants from PDL cells stimulated with (10 ng/mL) or without (0 ng/mL) IL-1b treatment (n = 9) Medium
Supernatant without IL-1b treatment Supernatant without IL-1b treatment + anti-MCP-1 Supernatant with IL-1b treatment Supernatant with IL-1b treatment + anti-MCP1 + anti-MCP-1
Mean
SD
95% confidence interval Lower boundary
Upper boundary
33.66
9.29
24.38
42.95
16.33
9.57
6.76
25.93
342.11
17.60
324.51
359.71
134.92*
13.69
121.23
148.61
*p < 0.01, compared with anti-MCP-1 antibody-free supernatant (10 ng/mL IL-1b treatment).
(a)
a
b
c
d
e
f
(b) numbers of migrated THP-1 cells
400 *
*
350 300 250
*
200 150 100
*
50 0 0 ng/ml
1 ng/ml
5 ng/ml
10 ng/ml
25 ng/ml
The concentration of IL-1β Fig. 3 The effect of THP-1 cell migration induced by different mediums. (A) THP-1 cell migration assay was performed in different groups: (a) negative control group, induced by the medium containing IL-1b; (b) control group, induced by the supernatant of PDL cells without IL-1b treatment; and (c–f) experimental groups, induced by supernatants from PDL cells stimulated with IL-1b (1–25 ng/mL). Migrated cells were stained with crystal violet. (B) The cell number was counted in five random fields (magnification bar: 50 lm). The data shown are the means SD of nine repeated experiments. By Student’s two-tailed t-test for unpaired comparisons, it is shown that the supernatants from PDL cells stimulated with IL-1b attracted more THP-1 cells than the supernatants from the cells in the control group did (*p < 0.05). 386
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MCP-1 expression in PDL cells induced by IL-1b Effect of PDL cells induced by IL-1b on THP-1 cell migration The means, standard deviations (SD) and 95% confidence intervals of the numbers of migrated THP-1 cell under different conditions are described in Tables 3 and 4. It was shown that the supernatants from PDL cells stimulated with IL-1b attracted more THP-1 cells (Fig. 3Ac–f) than those from PDL cells without IL-1b stimulation (Fig. 3Ab) (p < 0.05). When the supernatant from PDL cells was instead by the medium containing IL-1b in the negative control group, no significant attracting effect (p > 0.05)
(a)
DISCUSSION
a
b
c
d
(b) *
400 numbers of migrated THP-1 cells
was shown (Fig. 3Aa). It was suggested that IL-1b could not attract THP-1 cells directly, and that the attracting effect was from the PDL cells induced with IL-1b. In the blocking experiments, it was shown that the THP-1 cell migration induced by the supernatant could be blocked by the anti-MCP-1 antibody (Fig. 4). Compared with anti-MCP-1 antibody-free supernatant (Fig. 4Ad), THP-1 cell migration decreased significantly (p < 0.05) in the supernatant incubated with 1 lg/mL of anti-MCP-1 antibody (Fig. 4Ac). It was suggested that MCP-1 was one of the main chemokines for monocytes in the supernatant.
anti-MCP-1 antibody-free
350 300
anti-MCP-1 antibody
250 200 150 100 50 0
0 ng/ml
10 ng/ml The concentration of IL-1β Fig. 4 Blocking effects of anti-MCP-1 antibody on THP-1 cell migration. (A) An assay of THP-1 cell migration was performed using supernatants of PDL cells with (d) or without (b) 10 ng/mL of IL-1b treatment; THP-1 cell migration was blocked by 1 lg/mL of anti-MCP-1 antibody in the supernatants (a, c). (B) The migrated THP-1 cell number was counted in five random fields (magnification bar: 50 lm). The data shown are the means SD of nine repeated experiments. By Student’s two-tailed t-test for unpaired comparisons, it is shown that anti-MCP-1 antibody-free supernatants attracted more THP-1 cells than the antiMCP-1 antibody supernatants did (*p < 0.01). © 2015 Australian Dental Association
Orthodontic root resorption is mediated by an inflammatory reaction.22 The recruitment of mononuclear cells can be observed during the resorption process.19–21,28 Odontoclasts are needed for the resorption of the mineralized matrix of dental tissues. Odontoclasts are the same cell type as osteoclasts, and they are both called clastic cells.29–32 Mononuclear cells are derived from a monocyte–macrophage cell lineage. When mononuclear cells are attracted to certain mineralized surfaces, they fuse into clastic cells to exert their resorptive activity.32 Peripheral blood mononuclear cells may differentiate into osteoclastlike cells in the presence of RANKL plus M-CSF.30,33 Therefore, the mononuclear cells recruited at the site of root resorption may be regarded as precursors of odontoclasts. The mononuclear cells migrate from the peripheral blood and could be activated to differentiate into macrophages or fuse to osteoclasts/odontoclasts. It is important to determine the precise mechanism of mononuclear cell recruitment for dealing with orthodontic root resorption. During the orthodontic root resorption process, IL1b would increase in the PDL.1,2 MCP-1 has been identified as having strong chemotactic activity on monocytes,10–14 and plays an important role in the development of bone resorption.34 In this study, the expression of MCP-1 in PDL cells induced by IL-1b was investigated in vitro. The results indicated that the high concentrations of IL-1b (10 ng/mL and 25 ng/mL) could increase the mRNA and protein expression of MCP-1. However, lower concentrations of IL-1b did not show a significant effect on MCP-1 expression in PDL cells. This was consistent with the findings from previous studies that IL-1b can induce the expression of MCP-1 in chondrocytes and osteoblastic cells.16,17 THP-1 cells were widely used to investigate the regulatory mechanism of the monocyte–macrophage migration in relation to the pathophysiology of vas387
J Jin and J Cao cular inflammation and bone degeneration diseases.35,36 In this study, it was shown that the supernatant of PDL cells treated with IL-1b attracted more THP-1 monocytes in the migration assay than those of the cells without IL-1b treatment did. Many other chemokines and proinflammatory cytokines can be released by PDL cells under IL-1b stimulation.37–40 Furthermore, IL-1b and FBS might also be in the supernatant. When the supernatant of PDL cells was replaced by the DMEM medium supplemented containing FBS and IL-1b (in the negative control group), no significant attracting effect was shown. It was suggested that the attracting effect was from the PDL cells induced with IL-1b, and this effect was not due to the direct effects of IL-1b or FBS. In addition, in the blocking experiments, it was shown that the THP-1 cell migration induced by the supernatant could be blocked by the anti-MCP-1 antibody. Therefore, it was concluded that MCP-1 played a main role in THP-1 cell migration induced by IL-1b-stimulated PDL cells. This research implied that during the process of orthodontic root resorption, inflammatory cytokine IL1b could induce MCP-1 expression at a high level in PDL cells, and the monocytes were probably directed to migrate via MCP-1 signals through an IL-1b-dependent mechanism. Cementum metabolism is a complicated process in physiology, and is made even more complicated in pathology and disease. There might be many chemokines, cytokines, proteins, compounds and particular cell types involved in the processes of orthodontic root resorption. Since mononuclear cells could differentiate into macrophages or fuse to form odontoclasts, the recruitment of mononuclear cells may be one of the mechanisms underlying root resorption. Therefore, according to the results of this study, it might become a potential root resorption interruption way to inhibit the effect of IL-1b on the expression of MCP-1 in human PDL cells. Further studies are needed to illustrate the pathway of the upregulated expression of MCP-1, as induced by IL-1b. ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (No. 81070870). REFERENCES 1. Brezniak N, Wasserstein A. Orthodontically induced inflammatory root resorption. Part I: the basic science aspects. Angle Orthod 2002;72:175–179. 2. Weltman B, Vig KW, Fields HW, Shanker S, Kaizar EE. Root resorption associated with orthodontic tooth movement: a systematic review. Am J Orthod Dentofacial Orthop 2010;137: 462–476. 388
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MCP-1 expression in PDL cells induced by IL-1b 22. Casa MA, Faltin RM, Faltin K, Arana-Chavez VE. Root resorption on torqued human premolars shown by tartrate-resistant acid phosphatase histochemistry and transmission electron microscopy. Angle Orthod 2006;76:1015–1021. 23. Mavragani M, Amundsen OC, Selliseth NJ, Brudvik P, Selvig KA. Early root alterations after orthodontic force application studied by light and scanning electron microscopy. Eur J Orthod 2004;26:119–128. 24. Osdoby P, Martini MC, Caplan AI. Isolated osteoclasts and their presumed progenitor cells, the monocyte, in culture. J Exp Zool 1982;224:331–344. 25. Nicholson GC, Malakellis M, Collier FM, et al. Induction of osteoclasts from CD14-positive human peripheral blood mononuclear cells by receptor activator of nuclear factor kappaB ligand (RANKL). Clin Sci (Lond) 2000;99:133–140. 26. Sousa NG, Cardoso CR, Silva JS, Kuga MC, Tanomaru-Filho M, Faria G. Association of matrix metalloproteinase inducer (EMMPRIN) with the expression of matrix metalloproteinases1, -2 and -9 during periapical lesion development. Arch Oral Biol 2014;59:944–953. 27. Rossi M, Whitcomb S, Lindemann R. Interleukin-1 beta and tumor necrosis factor-alpha production by human monocytes cultured with L-thyroxine and thyrocalcitonin: relation to severe root shortening. Am J Orthod Dentofacial Orthop 1996;110:399–404. 28. Konermann A, Beyer M, Deschner J, et al. Human periodontal ligament cells facilitate leukocyte recruitment and are influenced in their immunomodulatory function by Th17 cytokine release. Cell Immunol 2012;272:137–143. 29. Arana-Chavez VE, Bradaschia-Correa V. Clastic cells: mineralized tissue resorption in health and disease. Int J Biochem Cell Biol 2009;41:446–450. 30. Udagawa N, Takahashi N, Akatsu T, et al. Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc Natl Acad Sci U S A 1990;87:7260–7264. 31. McHugh KP, Shen Z, Crotti TN, et al. Role of cell-matrix interactions in osteoclast differentiation. Adv Exp Med Biol 2007;602:107–111. 32. Lerner UH. Osteoclast formation and resorption. Matrix Biol 2000;19:107–120.
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33. Massey HM, Flanagan AM. Human osteoclasts derive from CD14-positive monocytes. Br J Haematol 1999;106: 167–170. 34. Silva TA, Garlet GP, Fukada SY, Silva JS, Cunha FQ. Chemokines in oral inflammatory diseases: apical periodontitis and periodontal disease. J Dent Res 2007;86:306–319. 35. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer 1980;26: 171–176. 36. Qin Z. The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature. Atherosclerosis 2012;221:2–11. 37. Mustafa M, Zarrough A, Bolstad AI, et al. Resolvin D1 protects periodontal ligament. Am J Physiol Cell Physiol 2013;305: C673–679. 38. Luo LJ, Liu F, Lin ZK, et al. Genistein regulates the IL-1 beta induced activation of MAPKs in human periodontal ligament cells through G protein-coupled receptor 30. Arch Biochem Biophys 2012;522:9–16. 39. Cao Z, Li C, Zhu G. Inhibitory effects of baicalin on IL-1betainduced MMP-1/TIMP-1 and its stimulated effect on collagen-I production in human periodontal ligament cells. Eur J Pharmacol 2010;641:1–6. 40. Rossa C Jr, Liu M, Patil C, Kirkwood KL. MKK3/6-p38 MAPK negatively regulates murine MMP-13 gene expression induced by IL-1beta and TNF-alpha in immortalized periodontal ligament fibroblasts. Matrix Biol 2005;24:478–488.
Address for correspondence: Professor Jun Cao State Key Laboratory of Military Stomatology Department of Orthodontics School of Stomatology The Fourth Military Medical University Xi’an 710032 China Email: [email protected]
389
The official journal of the Australian Dental Association
Australian Dental Journal 2015; 60: 382–389 doi: 10.1111/adj.12234
Upregulated expression of monocyte chemoattractant protein-1 in human periodontal ligament cells induced by interleukin-1b J Jin,* J Cao* *State Key Laboratory of Military Stomatology, Department of Orthodontics, School of Stomatology, the Fourth Military Medical University, Xi’an, China.
ABSTRACT Background: Root resorption during orthodontic treatment is a complex and sterile inflammatory process, characterized by the recruitment of mononuclear cells in the local periodontal ligament. This study aimed to investigate whether interleukin (IL)-1b could induce the migration of monocytes through upregulating monocyte chemoattractant protein (MCP)1 expression in human periodontal ligament cells. Methods: Human periodontal ligament cells were cultured in medium containing various IL-1b concentrations. After 24 hours of incubation, the messenger RNA (mRNA) and MCP-1 protein in periodontal ligament cells were detected by reverse transcriptase–polymerase chain reaction and Western blot analysis, respectively. The effect of supernatants of periodontal ligament cells on THP-1 cells was analysed via migration assay. Results: IL-1b (10 ng/mL and 25 ng/mL) increased the expression of MCP-1 mRNA and protein in periodontal ligament cells (p < 0.05). In addition, the supernatants from the IL-1b-treated periodontal ligament cells increased the migratory response of THP-1 cells (p < 0.05), which could be blocked by the anti-MCP-1 antibody. Conclusions: IL-1b had the potential to induce the migratory response of monocytes via upregulation of the expression of MCP-1 in human periodontal ligament cells and could contribute to orthodontic root resorption. Keywords: Chemotaxis, interleukin-1b, monocyte, monocyte chemoattractant protein-1, periodontal ligament cell. Abbreviations and acronyms: DMEM = Dulbecco’s Modified Eagle’s Medium; FBS = fetal bovine serum; IL = interleukin; MCP = monocyte chemoattractant protein; M-CSF = macrophage colony-stimulating factor; PCR = polymerase chain reaction; PDL = periodontal ligament; RANKL = receptor activator of nuclear factor kappa-B; RIPA = radioimmunoprecipitation assay; RT-PCR = reverse transcription–polymerase chain reaction. (Accepted for publication 2 October 2014.)
INTRODUCTION Root resorption, the unwanted side effect following orthodontic tooth movement, is triggered by a sterile inflammatory process.1–3 The inflammatory process is characterized by mononuclear cell recruitment. Chemokines and proinflammatory cytokines released by periodontal ligament (PDL) cells underlie the inflammatory state. Interleukin (IL)-1b is a proinflammatory cytokine and a potent stimulus for bone resorption and osteoclastic cell recruitment during orthodontic tooth movement.4 Diseases that affect the properties of bone often alter the metabolism of cementum since they have a similar composition.5 Increased levels of IL-1b have been found at the compression side of the PDL during orthodontic tooth movement.3,6,7 A combina382
tion of loading and an increased level of IL-1b has been discussed as being responsible for the loss of cementum.6 Human primary cementoblasts subjected to compression and IL-1 stimulation could impede some cementogenesis-associated proteins expression, such as bone sialoprotein and cementum-derived protein.7 A role for IL-1b in the pathogenesis of root resorption is suggested since the polymorphism of the IL-1b gene is associated with root resorption.4,8 During the root resorption process, the PDL cells are constantly stimulated by increasing IL-1b. Chemokines direct leukocyte trafficking and positioning within the tissues, and they play critical roles in regulating immune responses and inflammation. Chemokines are a large family of small proteins, which play the crucial role of chemotaxis in cell trafficking, angiogenesis, cell proliferation, apoptosis, tumour metastasis © 2015 Australian Dental Association
MCP-1 expression in PDL cells induced by IL-1b and host defense.9,10 As a potent chemokine for monocytes, monocyte chemoattractant protein (MCP)-1 is implicated in the pathogenesis of diseases characterized by monocyte infiltration.10–14 It was demonstrated that MCP-1 presented in the PDL cells in the root resorption area,15 which implied that monocytes were probably directed to migrate via signals from MCP-1, which was released from resident cells of PDL during the resorption process. It has been shown that IL-1b can induce MCP-1 expression in many resident cells, such as chondrocytes and osteoblastic cells.16,17 However, there is no direct evidence for the role of IL-1b in MCP-1 expression in PDL cells. A singular differentiation process within the haematopoietic system is represented by the differentiation of monocytes to osteoclasts. Osteoclasts are giant, multinucleated cells, and are specialized in degrading bone.18 Since cementum and bone have a similar composition, mononuclear cells are thought to play a role in root resorption.5 When stimulated with receptor activator of nuclear factor kappa-B (RANKL) and macrophage colony-stimulating factor (M-CSF), mononuclear cells could differentiate into macrophages or fuse to form odontoclasts.19–25 Additionally, factors produced by mononuclear cells may contribute to resorption or injury.26,27 Despite the potential importance of mononuclear cells in root resorption, the molecular mechanism involved in the recruitment of monocytes has been unclear, until now. The present study aimed to investigate the expression of MCP-1 in PDL cells induced by IL-1b, as well as the migration of THP-1 cells regulated by the expression of MCP-1. It was shown that IL-1b could increase the migratory response of monocytes via upregulation of the expression of MCP-1. This may be one of the mechanisms through which monocyte recruitment occurs during orthodontic root resorption. METHODS Cell culture PDL cells were obtained from 20 teeth of five patients (12–13 years of age) following premolar extraction, as indicated during orthodontic treatment. The research protocol was approved by the Human Experimentation Committee of the School of Stomatology, the Fourth Military Medical University, People’s Republic of China. Informed consent was obtained from the patients’ parents after being provided with an explanation of the study. PDL cells were cultured as described previously.28 Briefly, the PDL was gently scraped off from the middle third of the root surface to avoid contamination from the gingival and apical © 2015 Australian Dental Association
tissues. The tissue explants were transferred to cell culture flasks containing Dulbecco’s Modified Eagle’s Medium (DMEM; Gibcoâ BRL, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with antibiotics (penicillin 100 U/mL, streptomycin 100 lg/ mL) and 10% fetal bovine serum (FBS; Gibcoâ BRL, Thermo Fisher Scientific, Waltham, MA, USA). The flasks were placed in a water-jacketed cell/tissue incubator with 5% CO2. The cells were allowed to migrate from the explants. After reaching confluence, the cells were detached by incubation with 0.25% trypsin and reseeded at a density of 80 000 cells/mL. PDL cells were used between passages three and five for the experiments. Cell treatment First, all the PDL cells from the five patients were mixed. The mixed PDL cells were then seeded in six-well plates at 1 9 106 cells/well. After reaching 80% confluence, the cells were serum starved with serum-free DMEM overnight to achieve standardized conditions. In the experimental group, the PDL cells were treated with various concentrations (1 ng/mL, 5 ng/mL, 10 ng/mL or 25 ng/mL) of recombinant human IL-1b (Sigma-Aldrich Company, St. Louis, MO, USA) in 3 mL of DMEM supplemented with 1% FBS for 24 hours. In the control group, the PDL cells were incubated in 3 mL of DMEM supplemented with 1% FBS for 24 hours. The cells in different treatment groups were then collected and used for reverse transcription–polymerase chain reaction (RT-PCR) analysis and Western blot analysis. The cell-free supernatants were stored at 70 °C for the migration assay. Reverse transcription–polymerase chain reaction analysis Total RNA from the PDL cells was extracted using TRIzolâ reagent (Gibcoâ BRL, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The RNA samples were reversetranscribed into first-strand complementary DNA (cDNA) using the SuperScriptâ Preamplification System (Gibcoâ BRL, Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was amplified by polymerase chain reaction (PCR). PCR was carried out in 50 lL of PCR reaction buffer containing 2.5 lL of the template, 1.0 U of Taq polymerase (Promega Corporation, Fitchburg, WI, USA), 0.2 mM of deoxyribonucleotide triphosphates (dNTPs), and 100 pmol forward and reverse primers. The reaction mixture was incubated at 94 °C for 50 seconds. It was then subjected to 30 cycles of denaturing at 94 °C for 35 seconds, annealed at 58 °C for 60 seconds, and extended at 72 °C for 383
J Jin and J Cao 30 seconds. The reaction was performed in duplicate. The sequences of PCR primers for MCP-1 were 50 -CT TCTGTGCCTGCTGCTCATA-30 (forward) and 50 -CT TTGGGACACTTGCTGCTG-30 (reverse). Those for GAPDH, a housekeeping gene, were 50 -CGGAGTCA ACGGATTTGGTCGTAT-30 (forward) and 50 -AGCC TTCTCCATGGTGGTGAAGAC-30 (reverse). The PCR products were resolved on 1.8% agarose gel and visualized by ethidium bromide staining. The relative intensities of the PCR products were analysed with picture imaging software (Scion Image, Informer Technologies, Inc., Frederick, MD, USA). The results were normalized to the corresponding GAPDH messenger RNA (mRNA) levels. Western blot analysis PDL cells in each well were washed with PBS, lysed with ice cold radioimmunoprecipitation assay (RIPA) buffer (Bio-Rad Laboratories Inc., Hercules, CA, USA), and centrifuged at 10 000 g for 5 minutes at 4 °C. The supernatants were collected and subjected to Western blot analysis. The protein content of the samples was measured using the BCA protein assay kit according to the manufacturer’s protocol (Pierce Chemical Company; Thermo Fisher Scientific, Waltham, MA, USA). Each protein sample (20 lg) was subjected to sodium dodecylsulfate–polyacrylamide electrophoresis (SDS-PAGE) and transferred from SDS gels onto a poly-vinyliden difluoride (PDVF) membrane for immunoblot analysis. The membranes were blocked with 5% BSA in Tris buffered saline with Tweenâ (TBST). The membrane was then incubated at 4 °C for 8 hours with rabbit anti-human MCP-1 antibodies (Abcam plc, London, UK) diluted 1:500 in 5% non-fat dry milk in TBST. After washing, the membranes were incubated at room temperature for 1 hour with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (IgG) (GE, Arlington Heights, IL, USA) diluted 1:3000 in 5% non-fat dry milk in TBST, and developed using the ECL system (GE, Arlington Heights, IL, USA). b-actin-specific antibody (1:1000; Abcam, London, UK) was used for loading controls on the stripped membranes. The relative protein expressions were analysed with picture imaging software (Scion Image; Informer Technologies, Inc., Frederick, MD, USA). The results were normalized to the corresponding b-actin expression levels. Migration assay The THP-1 cell line, derived from human acute monocytic leukaemia, was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI 1640 (Gibcoâ BRL; 384
Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% FBS, 100 U/mL of penicillin, 100 lg/mL of streptomycin and 2 mM of L-glutamine. Cell migration was assessed in 24-well chemotaxis chambers fitted with 8.0 lm transwell membranes (Corning, Rosemont, VA, USA). Then, 1 9 106 THP-1 cells in 100 lL RPMI 1640 containing 1% FBS were added to the upper wells. In the experimental groups, the supernatants (150 lL) of PDL cells cultured in medium with various IL-1b concentrations (1 ng/mL, 5 ng/mL, 10 ng/mL and 25 ng/mL) were added to the lower chamber of the transwell system. In the control group, the supernatant (150 lL) of PDL cells cultured in medium without IL-1b (0 ng/mL) was added. In the negative control group, the medium containing IL-1b was used instead of the supernatant. The medium was DMEM supplemented with 1% FBS. In the blocking experiments, the assay of THP-1 cell migration was performed using supernatants of PDL cells with or without 10 ng/mL of IL-1b treatment. In the blocking groups, the supernatants of PDL cells were incubated with 1 lg/mL anti-MCP-1 antibody (Abcam, London, UK) overnight at 4 °C
Table 1. Effect of various IL-1b concentrations on relative mRNA expression (MCP-1/GAPDH) in PDL cells (n = 4) IL-1b concentration (ng/mL)
0 1 5 10 25
Mean
0.80 0.80 0.80 0.95* 0.94*
SD
0.03 0.05 0.02 0.03 0.04
95% confidence interval Lower boundary
Upper boundary
0.77 0.75 0.78 0.92 0.91
0.83 0.85 0.82 0.98 0.97
*p < 0.05, compared with the control group (0 ng/mL IL-1b treatment).
Table 2. Effect of various IL-1b concentrations on relative protein expression (MCP-1/b-actin) in PDL cells (n = 4) IL-1b concentration (ng/mL)
0 1 5 10 25
Mean
0.41 0.41 0.42 0.92* 0.91*
SD
0.04 0.02 0.05 0.03 0.04
95% confidence interval Lower boundary
Upper boundary
0.37 0.38 0.38 0.89 0.87
0.44 0.43 0.47 0.96 0.95
*p < 0.05, compared with the control group (0 ng/mL IL-1b treatment). © 2015 Australian Dental Association
MCP-1 expression in PDL cells induced by IL-1b (b) IL-1β (ng/ml) 0
1
5
10
25
500 bp 250 bp
MCP-1 (166 bp)
100 bp 500 bp
GAPDH (306 bp)
250 bp 100 bp
1.2
Relative mRNA expression (MCP-1/GAPDH)
(a)
*
*
1 0.8 0.6 0.4 0.2 0
0 ng/ml 1 ng/ml 5 ng/ml 10 ng/ml 25 ng/ml
The concentration of IL-1β
Fig. 1 Effects of IL-1b on the MCP-1 mRNA expression in PDL cells. (a) PDL cells were exposed to various IL-1b concentrations (1–25 ng/mL), and total RNA isolated from the cells treated at each concentration was subjected to RT-PCR analysis. (b) The expression of each molecule shown in (a) was quantified by image analysis. The results are presented as the ratio of the mRNA level to GAPDH in each molecule. The means SD from four repeated experiments are reported. By ANOVA with Bonferroni’s post hoc test, significant differences compared to the control group (0 ng/mL; IL-1b treatment) are shown (*p < 0.05).
(a)
(b)
0
1
5
10
25
MCP-1
β-actin
Relative protein expression (MCP-1/β-actin)
IL-1β (ng/ml)
1.2 *
1
*
0.8 0.6 0.4 0.2 0 0 ng/ml 1 ng/ml 5 ng/ml 10 ng/ml 25 ng/ml The concentration of IL-1β
Fig. 2 Effects of IL-1b on the MCP-1 protein expression in PDL cells. (a) PDL cells were exposed to various IL-1b concentrations (1–25 ng/mL), and the whole-cell lysates were extracted, resolved on 8% SDS-PAGE, and subjected to Western blot analysis with an antibody against MCP-1. (b) Data in the graph were shown as the means SD from four repeated experiments. By ANOVA with Bonferroni’s post hoc test, significant differences compared to the control group (0 ng/mL; IL-1b treatment) are shown (*p < 0.05).
before the migration assay. In the control groups, the supernatants were without the anti-MCP-1 treatment. The transwell membranes were removed after 90 minutes of incubation at 37 °C. Non-migrated cells were then removed from the system. The migrated cells were fixed in 4% formaldehyde for 15 minutes at 37 °C and stained with crystal violet. The cell number in five random fields (original magnification: 4009) was counted. Statistics The RT-PCR and Western blot experiments were repeated four times, and the migration assay was performed nine times. The data of the results in the repeated experiments followed a normal distribution. The means, standard deviations (SD) and 95% confidence intervals were calculated. Statistical significance was calculated using analysis of variance (ANOVA) and Student’s two-tailed t-test for unpaired comparisons with the Bonferroni test for post hoc analysis, as appropriate. P-values less than 0.05 were regarded as statistically significant. © 2015 Australian Dental Association
RESULTS Effect of IL-1b on MCP-1 expression in PDL cells The means, standard deviations (SD) and 95% confidence intervals of MCP-1 mRNA and protein expression under various IL-1b concentrations are
Table 3. Effect of PDL cells stimulated with various IL-1b concentrations on THP-1 cell migration (n = 9) IL-1b concentration (ng/mL) Mean
0 1 5 10 25
31.11 62.22* 193.22* 342.22* 352.66*
SD
5.30 7.27 6.62 5.64 7.58
95% confidence interval Lower boundary
Upper boundary
25.81 54.95 186.60 336.58 345.08
36.41 69.49 199.84 347.86 360.25
*p < 0.05, compared with the control group (0 ng/mL IL-1b treatment). 385
J Jin and J Cao described in Tables 1 and 2. As shown in Fig. 1, PDL cells expressed MCP-1 mRNA at basal levels without IL-1b stimulation. Treatment with IL-1b could increase the MCP-1 mRNA expression of PDL cells, with no effect at 1 ng/mL and 5 ng/mL (p > 0.05), and significant effects were observed at 10 ng/mL and 25 ng/mL (p < 0.05). Similar results were found for the MCP-1 protein by Western blot analysis (Fig. 2). Though the expression of MCP-1 protein in the PDL cells without IL-1b stimulation could be detected at low levels, IL-1b with higher concentrations (10 ng/mL and 25 ng/mL) increased the protein expression significantly (p < 0.05). IL-1b at lower concentrations (1 ng/mL and 5 ng/mL) could not significantly change the MCP-1 expression in PDL cells (p > 0.05). These results demonstrated a bioactive role of IL-1b in modulating the MCP-1 expression of PDL cells.
Table 4. The blocking effect of anti-MCP-1 on THP-1 cell migration induced by supernatants from PDL cells stimulated with (10 ng/mL) or without (0 ng/mL) IL-1b treatment (n = 9) Medium
Supernatant without IL-1b treatment Supernatant without IL-1b treatment + anti-MCP-1 Supernatant with IL-1b treatment Supernatant with IL-1b treatment + anti-MCP1 + anti-MCP-1
Mean
SD
95% confidence interval Lower boundary
Upper boundary
33.66
9.29
24.38
42.95
16.33
9.57
6.76
25.93
342.11
17.60
324.51
359.71
134.92*
13.69
121.23
148.61
*p < 0.01, compared with anti-MCP-1 antibody-free supernatant (10 ng/mL IL-1b treatment).
(a)
a
b
c
d
e
f
(b) numbers of migrated THP-1 cells
400 *
*
350 300 250
*
200 150 100
*
50 0 0 ng/ml
1 ng/ml
5 ng/ml
10 ng/ml
25 ng/ml
The concentration of IL-1β Fig. 3 The effect of THP-1 cell migration induced by different mediums. (A) THP-1 cell migration assay was performed in different groups: (a) negative control group, induced by the medium containing IL-1b; (b) control group, induced by the supernatant of PDL cells without IL-1b treatment; and (c–f) experimental groups, induced by supernatants from PDL cells stimulated with IL-1b (1–25 ng/mL). Migrated cells were stained with crystal violet. (B) The cell number was counted in five random fields (magnification bar: 50 lm). The data shown are the means SD of nine repeated experiments. By Student’s two-tailed t-test for unpaired comparisons, it is shown that the supernatants from PDL cells stimulated with IL-1b attracted more THP-1 cells than the supernatants from the cells in the control group did (*p < 0.05). 386
© 2015 Australian Dental Association
MCP-1 expression in PDL cells induced by IL-1b Effect of PDL cells induced by IL-1b on THP-1 cell migration The means, standard deviations (SD) and 95% confidence intervals of the numbers of migrated THP-1 cell under different conditions are described in Tables 3 and 4. It was shown that the supernatants from PDL cells stimulated with IL-1b attracted more THP-1 cells (Fig. 3Ac–f) than those from PDL cells without IL-1b stimulation (Fig. 3Ab) (p < 0.05). When the supernatant from PDL cells was instead by the medium containing IL-1b in the negative control group, no significant attracting effect (p > 0.05)
(a)
DISCUSSION
a
b
c
d
(b) *
400 numbers of migrated THP-1 cells
was shown (Fig. 3Aa). It was suggested that IL-1b could not attract THP-1 cells directly, and that the attracting effect was from the PDL cells induced with IL-1b. In the blocking experiments, it was shown that the THP-1 cell migration induced by the supernatant could be blocked by the anti-MCP-1 antibody (Fig. 4). Compared with anti-MCP-1 antibody-free supernatant (Fig. 4Ad), THP-1 cell migration decreased significantly (p < 0.05) in the supernatant incubated with 1 lg/mL of anti-MCP-1 antibody (Fig. 4Ac). It was suggested that MCP-1 was one of the main chemokines for monocytes in the supernatant.
anti-MCP-1 antibody-free
350 300
anti-MCP-1 antibody
250 200 150 100 50 0
0 ng/ml
10 ng/ml The concentration of IL-1β Fig. 4 Blocking effects of anti-MCP-1 antibody on THP-1 cell migration. (A) An assay of THP-1 cell migration was performed using supernatants of PDL cells with (d) or without (b) 10 ng/mL of IL-1b treatment; THP-1 cell migration was blocked by 1 lg/mL of anti-MCP-1 antibody in the supernatants (a, c). (B) The migrated THP-1 cell number was counted in five random fields (magnification bar: 50 lm). The data shown are the means SD of nine repeated experiments. By Student’s two-tailed t-test for unpaired comparisons, it is shown that anti-MCP-1 antibody-free supernatants attracted more THP-1 cells than the antiMCP-1 antibody supernatants did (*p < 0.01). © 2015 Australian Dental Association
Orthodontic root resorption is mediated by an inflammatory reaction.22 The recruitment of mononuclear cells can be observed during the resorption process.19–21,28 Odontoclasts are needed for the resorption of the mineralized matrix of dental tissues. Odontoclasts are the same cell type as osteoclasts, and they are both called clastic cells.29–32 Mononuclear cells are derived from a monocyte–macrophage cell lineage. When mononuclear cells are attracted to certain mineralized surfaces, they fuse into clastic cells to exert their resorptive activity.32 Peripheral blood mononuclear cells may differentiate into osteoclastlike cells in the presence of RANKL plus M-CSF.30,33 Therefore, the mononuclear cells recruited at the site of root resorption may be regarded as precursors of odontoclasts. The mononuclear cells migrate from the peripheral blood and could be activated to differentiate into macrophages or fuse to osteoclasts/odontoclasts. It is important to determine the precise mechanism of mononuclear cell recruitment for dealing with orthodontic root resorption. During the orthodontic root resorption process, IL1b would increase in the PDL.1,2 MCP-1 has been identified as having strong chemotactic activity on monocytes,10–14 and plays an important role in the development of bone resorption.34 In this study, the expression of MCP-1 in PDL cells induced by IL-1b was investigated in vitro. The results indicated that the high concentrations of IL-1b (10 ng/mL and 25 ng/mL) could increase the mRNA and protein expression of MCP-1. However, lower concentrations of IL-1b did not show a significant effect on MCP-1 expression in PDL cells. This was consistent with the findings from previous studies that IL-1b can induce the expression of MCP-1 in chondrocytes and osteoblastic cells.16,17 THP-1 cells were widely used to investigate the regulatory mechanism of the monocyte–macrophage migration in relation to the pathophysiology of vas387
J Jin and J Cao cular inflammation and bone degeneration diseases.35,36 In this study, it was shown that the supernatant of PDL cells treated with IL-1b attracted more THP-1 monocytes in the migration assay than those of the cells without IL-1b treatment did. Many other chemokines and proinflammatory cytokines can be released by PDL cells under IL-1b stimulation.37–40 Furthermore, IL-1b and FBS might also be in the supernatant. When the supernatant of PDL cells was replaced by the DMEM medium supplemented containing FBS and IL-1b (in the negative control group), no significant attracting effect was shown. It was suggested that the attracting effect was from the PDL cells induced with IL-1b, and this effect was not due to the direct effects of IL-1b or FBS. In addition, in the blocking experiments, it was shown that the THP-1 cell migration induced by the supernatant could be blocked by the anti-MCP-1 antibody. Therefore, it was concluded that MCP-1 played a main role in THP-1 cell migration induced by IL-1b-stimulated PDL cells. This research implied that during the process of orthodontic root resorption, inflammatory cytokine IL1b could induce MCP-1 expression at a high level in PDL cells, and the monocytes were probably directed to migrate via MCP-1 signals through an IL-1b-dependent mechanism. Cementum metabolism is a complicated process in physiology, and is made even more complicated in pathology and disease. There might be many chemokines, cytokines, proteins, compounds and particular cell types involved in the processes of orthodontic root resorption. Since mononuclear cells could differentiate into macrophages or fuse to form odontoclasts, the recruitment of mononuclear cells may be one of the mechanisms underlying root resorption. Therefore, according to the results of this study, it might become a potential root resorption interruption way to inhibit the effect of IL-1b on the expression of MCP-1 in human PDL cells. Further studies are needed to illustrate the pathway of the upregulated expression of MCP-1, as induced by IL-1b. ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (No. 81070870). REFERENCES 1. Brezniak N, Wasserstein A. Orthodontically induced inflammatory root resorption. Part I: the basic science aspects. Angle Orthod 2002;72:175–179. 2. Weltman B, Vig KW, Fields HW, Shanker S, Kaizar EE. Root resorption associated with orthodontic tooth movement: a systematic review. Am J Orthod Dentofacial Orthop 2010;137: 462–476. 388
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Address for correspondence: Professor Jun Cao State Key Laboratory of Military Stomatology Department of Orthodontics School of Stomatology The Fourth Military Medical University Xi’an 710032 China Email: [email protected]
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