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Biological

Estrogen Modulates Cytokine Expression in Human Periodontal Ligament Cells

L. Shu1, S.-M. Guan2, S.-M. Fu1, T. Guo1, M. Cao1 and Y. Ding1,*

1 Department of Orthodontics and
2 Department of Oral Biology, School of Stomatology, the Fourth Military Medical University, Xi’an, 710032, China

Correspondence: * corresponding author, yinding2005{at}yahoo.com.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although systemic bone loss accompanying estrogen deficiency has been proposed as a risk factor for periodontal disease in post-menopausal women, the mechanisms involved remain unclear. The objective of this study was to elucidate the potential bone-sparing effect of estrogen (17β-estradiol, E2) via modulation of inflammatory cytokine production in human periodontal ligament (hPDL) cells. E. coli lipopolysaccharide (LPS) increased the production of pro-inflammatory cytokines TNF-{alpha}, IL-1β, IL-6, and receptor activator of NF- B ligand (RANKL) by hPDL cells at both mRNA and protein levels. E2 treatment reversed the stimulatory effects of LPS on pro-inflammatory cytokine expression by hPDL cells. Moreover, E2 up-regulated osteoprotegerin (OPG) expression and therefore attenuated the reduction of the OPG vs. RANKL ratio. Our results suggested that estrogen may play a significant role in modulating periodontal tissue responses to LPS, and may exert its bone-sparing effects on periodontal tissues via altering the expression of inflammatory cytokines in hPDL cells.

Key Words: 17β-estradiol • cytokine • periodontal ligament cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Both osteoporosis and periodontal disease are bone-resorptive diseases. Estrogen deficiency is believed to be one of the major causes of post-menopausal osteoporosis. Osteoporosis is considered as one of the risk factors for periodontal disease and tooth loss (Baxter and Fattore, 1993; Mohammad et al., 1994; von Wowern et al., 1994; Reddy, 2002; Jeffcoat et al., 2003). Clinical observations in post-menopausal women have confirmed an increased prevalence of periodontal disease with lower estrogen levels, even when oral hygiene remained unchanged (Krall et al., 1994; Reinhardt et al., 1999). Animal experiments with ovariectomized (OVX) rats have also demonstrated that estrogen deficiency may result in low mineral density in the mandible (Kuroda et al., 2003), a thin cortex of the mandibular body (Miller et al., 1991), and resorption of alveolar bone (Elovic et al., 1995). Furthermore, several studies have suggested that estrogen may have an important role in chronic inflammatory periodontal diseases (Geurs et al., 2003; Guncu et al., 2005). However, the etiology of estrogen-associated periodontal diseases remains an enigma, partly because the precise effects of estrogen on periodontal tissues at the molecular level are not yet known.

Periodontitis is a chronic inflammatory disease characterized by gingival inflammation and alveolar bone resorption. It is generally accepted that much of the periodontal tissue destruction observed in periodontal disease is host-mediated through inflammatory cytokines by local tissues and immune cells in response to the bacterial flora and its products/metabolites, especially lipopolysaccharide (LPS) (Quintero et al., 1995; Darveau et al., 1997). The periodontal ligament (PDL), which lies between alveolar bone and cementum, plays a vital role in maintaining the homeostasis of periodontal tissues by affecting coordinated balance of bone-forming and bone-resorbing activities (Lekic and McCulloch, 1996; Shimizu et al., 1996).

A recent study demonstrated that estrogen deficiency can provoke an imbalance in the remodeling sequence of periodontal tissues (Lerner, 2006b). Previous studies have focused mainly on whether estrogen affects the bone-forming capability of PDL cells, such as the production of osteocalcin (Morishita et al., 1998, 1999). However, the effects of estrogen on bone resorption in the periodontium have been less explored. Estrogen-deficiency-triggered changes in inflammatory cytokines are emerging as a common theme that may have a significant impact on bone resorption in periodontal tissues (Pfeilschifter et al., 2002). The inflammatory cytokines that have attracted the most attention are TNF-{alpha}, IL-1β, IL-6, OPG, and RANKL (Rickard et al., 1992; Rogers and Eastell, 2001; Pfeilschifter et al., 2002).

The present study was designed to explore the modulatory effect of estrogen on the bone-resorbing cytokines such as TNF-{alpha}, IL-1β, IL-6, and RANKL and the anti-resorptive factor OPG in hPDL cells, and to elucidate the potential bone-sparing effects of estrogen in periodontal tissues via altering the expression of inflammatory cytokines.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
Healthy hPDL tissue was obtained from the extracted (for orthodontic reasons) premolars of two females (13 and 15 years old). Both girls gave informed consent before tooth extraction. Ethical approval had been obtained from the Ethics Committee of the School of Stomatology, the Fourth Military Medical University, China. The hPDL cells were obtained according to the method of Somerman et al.(1988). The cells were cultured in phenol red-free Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% charcoal-stripped serum (GIBCO BRL, Rockville, MD, USA), 100 U/mL of penicillin, and 100 µg/mL of streptomycin at 37° C in a 5% CO2 humidified atmosphere. After reaching confluence, the cells were subcultured. Cells used in this study were from passages 4 to 8.

Treatment of hPDL Cells with E. coli LPS and E2
The hPDL cells were seeded in six-well plates at a density of 1 x 106 cells per well and were allowed to attach for 12 hrs. The cells were then silenced with serum-free DMEM overnight. They were then stimulated by E. coli LPS (Sigma, St. Louis, MO, USA; 20 µg/mL), with or without estrogen (17β-estradiol, E2, Sigma) (10–7, 10–8, or 10–9 M) for 6, 12, 24, 48, and 72 hrs. The untreated hPDL cells served as a normal control. To confirm the specificity of the estrogen effects, we used ICI 182,780 (Tocris Cookson, Ellisville, MO, USA; 10–6 M), a specific estrogen receptor antagonist, as negative control. At specific times, culture media were collected. The hPDL cells of all treatment groups underwent lysis in 0.2% Triton X-100, and the cell lysates were collected and subjected to centrifugation. The cell-free supernatants were stored at –70° C prior to assay.

Inflammatory Cytokine Measurement by ELISA
Secreted TNF-{alpha}, IL-6, IL-1β, OPG, and soluble RANKL (sRANKL) in conditioned culture media, and intracellular TNF-{alpha}, IL-6, IL-1β, and membrane-bound RANKL in the cell lysates were measured by commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s protocols. All experiments were performed in triplicate.

mRNA Expression Detection by Quantitative Real-time PCR and RT-PCR
Total RNA was extracted with Trizol reagent (GIBCO BRL) and was quantified spectrophotometrically. cDNA was synthesized with 2 µg RNA in a 20-µL reaction mixture containing 2.5 µM random hexamers and Superscript II reverse transcriptase (GIBCO BRL). Primer sequences of each gene (human TNF-{alpha}, IL-6, IL-1β, RANKL, OPG, and GAPDH) were described previously (Lagoo-Deenadayalan et al., 1993; Fukushima et al., 2003). PCR was conducted with 2 µL of the reverse transcript. Each PCR was initially performed in a thermal cycler (Biometra, Göttingen, Germany) with standardized amplification programs. The PCR products were subjected to electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining. Human GAPDH primers were used as internal standards. PCR reactions were analyzed with picture-imaging software (Scion Image, Frederick, MD, USA). To quantify mRNA expression of OPG and RANKL, we performed real-time PCR as described, in the LightCycler system (Roche Diagnostics Co., Mannheim, Germany), with a SYBR Green reagent. Reaction products were quantified (Roche Quantification Software, Roche Diagnostics) with GAPDH as the reference gene.

Data Analysis
Data were presented as means ± SEM. All data were subjected to analysis of variance (ANOVA), followed by Scheffé’s correction for post hoc t test comparisons. P-values < 0.05 were considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of E2 on LPS-stimulated TNF-{alpha}, IL-1β, and IL-6 Expression in hPDL Cells
To investigate whether estrogen could affect spontaneous and LPS-stimulated inflammatory cytokine expression in hPDL cells, we measured TNF-{alpha}, IL-1β, and IL-6 expression at both mRNA and protein levels. A six-hour exposure to LPS significantly increased the secretion of TNF-{alpha}, IL-1β, and IL-6 by hPDL cells (P < 0.01, Fig. 1Go). The addition of E2 at physiological concentrations of 10–9 to 10–7 M to the culture media dose-dependently inhibited the stimulatory effects of LPS on inflammatory cytokine secretion. At 10–9 M, E2 only slightly decreased the effects of LPS; whereas, when 10–7 M of E2 was included in the culture media, the stimulatory effects of LPS were almost completely reversed. However, in the absence of LPS, E2 (10–7M) had little effect on inflammatory cytokine expression in hPDL cells. Pre-treatment of hPDL cells with the estrogen receptor antagonist ICI 182,780 resulted in a reversal of the influence of E2 on cytokine production. Therefore, a bioactive role for estrogen in modulating the responses of hPDL cells to LPS via inhibiting IL-1β, IL-6, and TNF-{alpha} production was demonstrated.


Figure 1
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  		Figure 1. Dose-dependent effects of 17β-estradiol on cytokine secretion in hPDL cells. The hPDL cells were stimulated by E. coli LPS (20 µg/mL) with or without different concentrations of 17β-estradiol from 10–9 M to 10–7 M for 6 hrs. (a) TNF-{alpha}. (b) IL-1β. (c) IL-6. Data were presented as means ± SEM (n = 12). **P < 0.01 vs. the control group. #P < 0.05, ##P < 0.01 vs. the LPS-treated group. {diamondsuit}P < 0.05, {diamondsuit}{diamondsuit}P < 0.01 vs. the LPS+E2 group.

 
The time-course effects of LPS and E2 on cytokine secretion were analyzed (Fig. 2AGo). Within 24 hrs of treatment, LPS dependently up-regulated TNF-{alpha}, IL-1β, and IL-6 secretion times. The levels of these cytokines remained constant between 24 and 48 hrs. Though E2 did not decrease cytokine concentrations to control levels, it greatly inhibited the LPS-induced cytokine up-regulation. For further exploration of the effect of E2 on cytokine production, the intracellular levels of TNF-{alpha}, IL-1β, and IL-6 were also evaluated (Fig. 2BGo). Within the first 6 hrs, LPS transiently increased intracellular TNF-{alpha}, IL-1β, and IL-6 levels vs. those in the control group. E2 alone had little effect on the intracellular cytokine levels, whereas co-treatment of E2 abrogated the up-regulatory effect of LPS at 6 hrs.


Figure 2
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  		Figure 2. Time-course effects of 17β-estradiol on cytokine production in hPDL cells. The hPDL cells were stimulated by E. coli LPS (20 µg/mL) with or without 17β-estradiol (10–7 M) for 6, 12, 24, or 48 hrs. (A) Secreted cytokine concentration in conditioned medium. (B) Intracellular cytokine level in cell lysates. (a) TNF-{alpha}. (b) IL-1β. (c) IL-6. Data were presented as means ± SEM (n = 12). *P < 0.05, **P < 0.01 vs. the control group. #P < 0.05, ##P < 0.01 vs. the LPS-treated group. {diamondsuit}P < 0.05, {diamondsuit}{diamondsuit}P < 0.01 vs. the LPS+E2 group.

 
To determine the effects of E2 on mRNA expression of TNF-{alpha}, IL-1β, and IL-6, we performed semi-quantitative RT-PCR. As evidenced by RT-PCR bands (Fig. 3Go), within the first 6 hrs, LPS significantly stimulated mRNA expression of TNF-{alpha}, IL-1β, and IL-6 vs. the control group, whereas E2 abrogated the up-regulation of mRNA expression. Furthermore, no changes in TNF-{alpha}, IL-1β, and IL-6 expression were detected in hPDL cells incubated with E2 alone.


Figure 3
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  		Figure 3. Changes of TNF-{alpha}, IL-1β, and IL-6 mRNA expression in hPDL cells at 6 hrs. The hPDL cells were stimulated by E. coli LPS (20 µg/mL) with or without 17β-estradiol (10–7 M) for 6 hrs. Steady-state mRNA levels were investigated by semi-quantitative RT-PCR analysis (40 cycles), and results were normalized to GAPDH. Results from the control group were arbitrarily set at 1, and each Fig. is representative of 3 separate experiments. *P < 0.05, **P < 0.01 vs. the control group. #P < 0.05 vs. the LPS-treated group. {diamondsuit}P < 0.05, {diamondsuit}{diamondsuit}P < 0.01 vs. the LPS+E2 group.

 
Effects of E2 on LPS-stimulated OPG and RANKL Expression in hPDL Cells
Since OPG and RANKL are another two important inflammatory cytokines, we next explored the effects of estrogen on OPG and RANKL expression in hPDL cells. Real-time PCR analysis (Fig. 4AGo) indicated that exposure of hPDL cells to LPS for 12 hrs had no effect on OPG and RANKL expression. E2 alone slightly increased OPG mRNA expression in comparison with the control group within 12 hrs, and the up-regulatory effect of E2 on OPG was observed up to 48 hrs. At the 24-hour treatment, LPS slightly promoted OPG and RANKL expression. However, at 48 hrs, LPS significantly enhanced RANKL expression, whereas co-treatment with E2 greatly attenuated the RANKL-inducing effects of LPS (P < 0.05, Fig. 4A-bGo) and significantly up-regulated the mRNA expression of OPG (P < 0.05, Fig. 4A-aGo). As a consequence, the OPG/RANKL ratio, which is a predictor of periodontal homeostasis, in the LPS+E2 group was significantly higher than that in the LPS group (P < 0.01, Fig. 4A-cGo).


Figure 4
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  		Figure 4. Effects of 17β-estradiol on OPG and RANKL expression in hPDL cells. (A) Both OPG and RANKL mRNA were subjected to real-time PCR analysis after cells were stimulated by LPS (20 µg/mL) with or without 17β-estradiol (10–7 M) for 12, 24, or 48 hrs. (a) OPG mRNA. (b) RANKL mRNA. (c) Ratio of OPG/RANKL mRNA. (B) Secreted OPG and RANKL in conditioned culture media and membrane-bound RANKL in the cell lysates were measured by ELISA at 24, 48, and 72 hrs. (a) Secreted OPG. (b) Membrane-bound RANKL. (c) Soluble RANKL. Data were presented as means ± SEM (n = 12). *P < 0.05, **P < 0.01 vs. the control group. #P < 0.05, ##P < 0.01 vs. the LPS-treated group.

 
To assess if the effects of estrogen on LPS-stimulated OPG and RANKL mRNA expression may result in parallel changes of protein synthesis, we examined the amounts of OPG and RANKL by ELISA (Fig. 4BGo). Since RANKL exists in both membrane-bound and soluble forms, we detected RANKL protein levels in both conditioned media and cell lysates. Within 24 hrs of E2 treatment, OPG secretion was up-regulated and remained up-regulated until 72 hrs, whereas no change of both membrane-bound and soluble RANKL was observed within 72 hrs. After treatment for 48 and 72 hrs, LPS slightly induced OPG secretion and significantly promoted RANKL synthesis simultaneously. Furthermore, the LPS-induced RANKL up-regulation was abrogated by E2, though spontaneous RANKL production was hardly affected by E2 alone (Fig. 4BGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A recent study demonstrated that the increased alveolar bone resorption induced by estrogen deficiency in OVX rat model is due to an increase of osteoclastogenesis (Kawamoto et al., 2002). We propose that estrogen may suppress osteoclastogenesis via the modulation of cytokine expression in the microenvironment of periodontal tissues.

In our study, it was found that E2 had little effect on the spontaneous expression of TNF-{alpha}, IL-1β, and IL-6 by hPDL cells. To explore the modulatory effects of E2 on the bone-resorbing cytokines in hPDL cells, we used E. coli LPS as a stimulant to induce the synthesis of inflammatory cytokines. Our results showed that E. coli LPS can induce the production of TNF-{alpha}, IL-1β, and IL-6 by hPDL cells, whereas co-treatment of E2 significantly suppressed LPS-stimulated production of TNF-{alpha}, IL-1β, and IL-6 in hPDL cells. This suggested that E2 may not alter the ability of hPDL cells to produce pro-inflammatory cytokines, but that it may modify the stimulatory effect of LPS on pro-inflammatory cytokines in hPDL cells. The inhibitory effect of E2 on LPS-induced TNF-{alpha}, IL-1β, and IL-6 synthesis could, at least in part, explain how E2 down-regulates osteoclastogenesis, for all these cytokines are thought to play important roles in osteoclastogenesis and may cause inflammatory resorption of alveolar bone by various mechanisms (Kobayashi et al., 2000).

Since the discovery of RANKL and its decoy receptor OPG, it has been believed that the ratio of RANKL/OPG determines osteoclast differentiation and activation, and that these two factors have emerged as essential mediators in the modulation of bone resorption (Hofbauer et al., 2000; Crotti et al., 2003). In this study, we observed that LPS up-regulated RANKL as well as OPG expression, although the OPG-inducing effect was not significant. We also found that E2 time-dependently increased OPG expression and attenuated the RANKL-inducing effects of LPS; however, no detectable effect on RANKL expression was observed with treatment of E2 alone. The LPS-induced decrease of OPG/RANKL ratio in hPDL cells was completely reversed by E2. These observations suggested that E2 may influence the progression of periodontal disease via altering the ratio of OPG to RANKL in hPDL cells.

A recent study suggested that E. coli LPS stimulated RANKL expression in hPDL cells through the prior induction of TNF-{alpha} and IL-1β (Wada et al., 2004). In our study, we found that it took 48 hrs to detect a significant change of LPS-induced RANKL expression, whereas TNF-{alpha}, IL-1β, and IL-6 expression was significantly enhanced within 6 hrs of LPS treatment. To verify whether the observed OPG and RANKL induction was mediated by upstream cytokines TNF-{alpha}, IL-1β, and IL-6, we added neutralizing antibodies for these cytokines. Our results demonstrated that neutralizing antibodies for these cytokines effectively inhibited LPS-induced OPG and RANKL expression, while they did not abolish the induction of OPG by E2 (data not shown). Therefore, we speculate that estrogen may modulate OPG expression in a manner independent of TNF-{alpha}, IL-1β, and IL-6, although LPS may induce OPG and RANKL expression in a manner dependent on upstream inflammatory stimulators. Although the molecular basis of estrogen’s effect on OPG expression in hPDL cells is not fully elucidated, it is possible that other cytokines, such as TGF-β, may contribute to the OPG-inducing effect of estrogen, either directly or indirectly (Takai et al., 1998).

In conclusion, this study showed an important anti-inflammatory effect of E2 in hPDL cells in vitro. We believe that, apart from the direct effect of E2 on osteoclasts, E2 may suppress osteoclastogenesis indirectly via modulating the synthesis of cytokines locally produced by hPDL cells, which may be one of the ways that E2 contributes to its anti-resorptive effect in periodontal tissue. In post-menopausal women, when the level of circulating estrogen declines, hPDL cells may produce inflammatory cytokines more readily in response to local stimulatory factors, such as LPS (reviewed by Lerner, 2006a,b). It is possible that estrogen affects the normal balance of bone formation and bone resorption by modulating the production of inflammatory cytokines in PDL cells. Further studies are necessary to investigate the exact mechanisms of estrogen on inflammatory cytokines in PDL cells at cellular and molecular levels.


   ACKNOWLEDGMENTS
 
This work was supported by the National Natural Science Foundation of China (No. 30572069).

Received for publication March 4, 2007. Revision received September 16, 2007. Accepted for publication October 17, 2007.


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 ABSTRACT
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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Dental Research, Vol. 87, No. 2, 142-147 (2008)
DOI: 10.1177/154405910808700214
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