Low magnitude of tensile strain inhibits IL-1beta-dependent induction of pro-inflammatory cytokines and induces synthesis of IL-10 in human periodontal ligament cells in vitro

Abstract

Applied mechanical loading induces inflammation in the periodontal ligament (PDL). However, the mechanisms involved in bone deposition at tension sites in an inflammatory environment are not clear. Here, in an in vitro model system, we show that equibiaxial tensile strain of low magnitude (TENS) provokes potent anti-inflammatory signals in PDL cells. TENS inhibits IL-1beta-induced synthesis of IL-1beta, IL-6, and IL-8 by inhibiting their mRNA expression, and thus significantly suppresses the amplification of IL-1beta-induced inflammatory responses in PDL cells. Additionally, as an anti-inflammatory signal, TENS induces IL-10 synthesis in the presence and absence of IL-1beta. These observations are the first to demonstrate that TENS antagonizes IL-1beta actions on PDL cells by (i) inhibiting IL-1beta-induced transcriptional regulation of proinflammatory cytokines, and (ii) inducing synthesis of IL-10, which may post-transcriptionally suppress the synthesis of pro-inflammatory cytokines.

Figure 1

Effects of various magnitudes of TENS on autoregulation of IL-1β in PDL cells. (A) Semiquantitative densitometric analysis of RT/PCR products for IL-1β following exposure of PDL cells to various magnitudes of TENS for 24 hrs. (B) Quantitative analysis of total IL-1β synthesis by ELISA in culture supernatants from PDL cells treated identically as in (A). The activating concentration of IL-1β (1 ng/mL) was subtracted from total IL-1β values. Results represent PL150 cells. Similar results were obtained from PL442, PL484, and PL75 cells. * indicates significant difference by ANOVA as compared with cells treated with IL-1β alone in (A) and between the two treatments under each horizontal bar in (B), calculated by ANOVA.

Figure 2

(A) PDL cells were either untreated (−) or treated (+) with 6% TENS alone, IL-1β (1 ng/mL), or TENS (6%) and IL-1β for 4, 24, or 48 hrs. Subsequently, mRNA was extracted and analyzed by RT/PCR for IL-1β induced IL-1β, TNFα, IL-6, and IL-8. Semiquantitative densitometric analysis of ethidium-bromide-stained RT/PCR products showed a significant reduction (p ≤ 0.05) in gene expression for each cytokine in cells treated with IL-1β and 6% TENS, in comparison with cells treated with IL-1β alone, at all time points. Analysis of (B) IL-1β, (C) IL-6, and (D) IL-8 synthesis in the culture supernatants of PDL cells as treated in (A) for 24 or 48 hrs. In (A), results are from PL150 cells performed in triplicate; similar results were obtained from PL442 and PL75 cells. In (B), (C), and (D), each point represents the mean and SEM of triplicate values from PL150 cells. Similar results were obtained from PL442 and PL75 cells. * indicates significant (p ≤ 0.05) inhibition by ANOVYA as compared with cells treated with IL-1β alone.

Figure 3

Effects of TENS on IL-10 mRNA expression and synthesis. (A) Ethidium-bromide-stained RT/PCR products for IL-10 after treatment of PDL cells as described in the legend to Fig. 2A, for 4, 24, or 48 hrs. In comparison with untreated (−) controls and cells treated (+) with IL-β, there is a distinct expression of IL-10 mRNA in cells treated with TENS alone and with IL-1β and TENS. (B) Semiquantitative densitometric analysis of RT/PCR products shown in (A), showing relative expression of IL-10 mRNA in cells treated with TENS alone and those treated with TENS and IL-β. (C) IL-10 synthesis as measured by ELISA in the culture supernatants of PDL cells as treated in (A) for 24 or 48 hrs. In (A) and (B), results represent PL150 cells performed in triplicate; similar results were obtained with PL442 and PL75 cells, and in (C), each point represents the mean and SEM of triplicate values in PL150 cells. Similar results were obtained from PL442 and PL75 cells. * indicates significant (p < 0.05) induction of IL-10 as compared with cells treated with IL-β alone, calculated by ANOVA.

Figure 4

(A)Ethidium-bromide-stained RT/PCR products for IL-1β in PL150 cells subjected to (a) IL-1β (1 ng/mL) alone, (b) IL-1β and TENS simultaneously, (c) pre-exposed to TENS for 1 hr prior to the addition of IL-1β (1 ng/mL), or (d) no treatment. Following a four-hour incubation, RNA was extracted and analyzed for IL-1β mRNA expression. The IL-1β;-induced IL-1β mRNA expression in cells pre-treated with TENS was similar to that in those treated with IL-1β alone. Similar results were obtained in PL75 cells. (B) Synthesis of IL-1β as measured by ELISA in cells treated for 24 hrs. Each point represents the mean and SEM of triplicate values. * indicates a significant (p ≤ 0.05) difference in comparison with cells treated with IL-1β alone.