Cyclic tensile strain acts as an antagonist of IL-1 beta actions in chondrocytes

Abstract

Inflammatory cytokines play a major role in cartilage destruction in diseases such as osteoarthritis and rheumatoid arthritis. Because physical therapies such as continuous passive motion yield beneficial effects on inflamed joints, we examined the intracellular mechanisms of mechanical strain-mediated actions in chondrocytes. By simulating the effects of continuous passive motion with cyclic tensile strain (CTS) on chondrocytes in vitro, we show that CTS is a potent antagonist of IL-1 beta actions and acts as both an anti-inflammatory and a reparative signal. Low magnitude CTS suppresses IL-1 beta-induced mRNA expression of multiple proteins involved in catabolic responses, such as inducible NO synthase, cyclo-oxygenase II, and collagenase. CTS also counteracts cartilage degradation by augmenting mRNA expression for tissue inhibitor of metalloproteases and collagen type II that are inhibited by IL-1 beta. Additionally, CTS augments the reparative process via hyperinduction of aggrecan mRNA expression and abrogation of IL-1 beta-induced suppression of proteoglycan synthesis. Nonetheless, the presence of an inflammatory signal is a prerequisite for the observed CTS actions, as exposure of chondrocytes to CTS alone has little effect on these parameters. Functional analysis suggests that CTS-mediated anti-inflammatory actions are not mediated by IL-1R down-regulation. Moreover, as an effective antagonist of IL-1 beta, the actions of CTS may involve disruption/regulation of signal transduction cascade of IL-1 beta upstream of mRNA transcription. These observations are the first to show that CTS directly acts as an anti-inflammatory signal on chondrocytes and provide a molecular basis for its actions.

FIGURE 1

Effects of various intensities of equibiaxial CTS on rhIL-1β-induced iNOS mRNA expression and NO production. A, Inhibition of rhIL-1β-induced iNOS mRNA expression in chondrocytes subjected to 2.5, 3.75, 5, 6.25, or 7.5% equibiaxial CTS at a rate of 0.05 Hz, for 24 h. The inhibition of mRNA expression was assessed by RT-QCPCR. B, Inhibition of rhIL-1β-dependent NO production in chondrocytes following the treatment described in A. NO production was assessed in the culture supernatants of the chondrocytes as total nitrite by Griess reaction (8). Control unstressed chondrocytes or chondrocytes treated with CTS alone did not exhibit iNOS mRNA expression or NO production. Each point in both figures represents the mean ± SEM of triplicate values. *, p ≤ 0.05 compared with cells treated with IL-1β alone.

FIGURE 2

Inhibition of rhIL-1β-dependent COX-II mRNA expression and PGE₂ synthesis by CTS in articular chondrocytes. A, COX-II mRNA expression in chondrocytes either untreated or subjected to rhIL-1β, CTS, or rhIL-1β and CTS for 4 or 24 h. B, PGE₂ synthesis by chondrocytes subjected to the treatment regimens described in A for 4, 24, or 48 h. PGE₂ synthesis was measured in the culture supernatants of chondrocytes by RIA. The data in A represent one of three separate experiments. The data in B represent the mean and SEM of triplicate values. *, p < 0.05 compared with cells treated with IL-1β alone.

FIGURE 3

Inhibition of rhIL-1β-dependent collagenase mRNA expression and synthesis by CTS in articular chondrocytes. A, Expression of collagenase mRNA in chondrocytes either untreated or subjected to rhIL-1β, CTS, or rhIL-1β and CTS for 4 or 24 h. B, Collagenase synthesis by chondrocytes subjected to the treatment regimens described in A. Collagenase synthesis was measured as the relative intensity of each band in Western blot analysis. The data in A are representative of one of three separate experiments. The data in B represent the mean and SEM of triplicate values. *, p < 0.05 compared with cells treated with IL-1β alone.

FIGURE 4

Effect of CTS on rhIL-1β-dependent inhibition of mRNA expression for TIMPs and type II collagen. Expression of mRNA for TIMP-I and TIMP-II (A) and type II collagen (B) in chondrocytes either untreated or exposed to rhIL-1β, CTS, or rhIL-1β and CTS. The mRNA expression was assessed by RT-PCR using 1 μg of RNA from cells in each group. Amplification of GAPDH mRNA was used to assure equal input in all lanes. The data represent one of three separate experiments. Bars represent the mean and SEM of triplicate values. *, p < 0.05 compared with cells treated with IL-1β alone.

FIGURE 5

Effect of CTS on rhIL-1β-dependent inhibition of proteoglycan synthesis. A, Expression of mRNA for aggrecan, biglycan, and versican in chondrocytes either untreated or exposed to rhIL-1β, CTS, or rhIL-1β and CTS for 4 or 24 h. The mRNA expression was assessed by RT-PCR. Amplification of GAPDH mRNA in 1 μg of total RNA was used as a standard. B, Quantitative assessment of aggrecan mRNA expression by RT-QCPCR in chondrocytes either untreated (■) or exposed to CTS (□), rhIL-1β (●), or CTS and rhIL-1β (○). C, Total chondroitin sulfate proteoglycans in the cell culture supernatants of chondrocytes shown in B. Proteoglycans were assessed by incorporation of Na₂³⁵SO₄ during the last 8 h of incubation. The data represent one of three separate experiments. Data in B and C are the mean ± SEM of triplicate values. *, p ≤ 0.05 compared with cells treated with rhIL-1B alone.

FIGURE 6

Suppression of rhIL-1β-dependent iNOS mRNA expression and NO production in chondrocytes exposed to CTS 1 h before or simultaneously with the addition of rhIL-1β. A, Expression of iNOS mRNA in chondrocytes exposed to CTS either 1 h before addition of rhIL-1β (-1 h) or simultaneously with rhIL-1β. mRNA expression was assessed by RT-PCR, 24 h after the addition of IL-1β. The data represent densitometric analysis of RT-PCR products for iNOS mRNA in one of three separate experiments. B, Analysis of NO production in the supernatants of the same chondrocytes exposed to the treatments shown in A, The data represent the mean and SEM of triplicate values. *, p ≤ 0.05 compared with cells treated with rhIL-1β and CTS.

FIGURE 7

Suppression of rhIL-1β-dependent iNOS mRNA expression and NO production in chondrocytes exposed to CTS at various time intervals following addition of rhIL-1β. A, Expression of iNOS mRNA in chondrocytes exposed to CTS either simultaneously with (0 h) or 1, 2, 4, or 8 h after the addition of rhIL-1β. iNOS mRNA expression was assessed by RT-PCR, 24 h after the addition of IL-1β. The data represent densitometric analysis of RT-PCR products for iNOS mRNA in one of three separate experiments. B, Analysis of NO production in the supernatants of chondrocytes subjected to the treatments shown in A. The data represent the mean and SEM of triplicate values. *, p ≤ 0.05 compared with cells treated with rhIL-1β alone.