Biomechanical signals exert sustained attenuation of proinflammatory gene induction in articular chondrocytes

Abstract

Objectives: Physical therapies are commonly used for limiting joint inflammation. To gain insight into their mechanisms of actions for optimal usage, we examined persistence of mechanical signals generated by cyclic tensile strain (CTS) in chondrocytes, in vitro. We hypothesized that mechanical signals induce anti-inflammatory and anabolic responses that are sustained over extended periods.

Methods: Articular chondrocytes obtained from rats were subjected to CTS for various time intervals followed by a period of rest, in the presence of interleukin-1beta (IL-1beta). The induction for cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP)-9, MMP-13 and aggrecan was analyzed by real-time polymerase chain reaction (PCR), Western blot analysis and immunofluorescence.

Results: Exposure of chondrocytes to constant CTS (3% CTS at 0.25 Hz) for 4-24 h blocked more than 90% (P<0.05) of the IL-1beta-induced transcriptional activation of proinflammatory genes, like iNOS, COX-2, MMP-9 and MMP-13, and abrogated inhibition of aggrecan synthesis. CTS exposure for 4, 8, 12, 16, or 20 h followed by a rest for 20, 16, 12, 8 or 4h, respectively, revealed that 8h of CTS optimally blocked (P<0.05) IL-1beta-induced proinflammatory gene induction for ensuing 16 h. However, CTS for 8h was not sufficient to inhibit iNOS expression for ensuing 28 or 40 h.

Conclusions: Data suggest that constant application of CTS blocks IL-1beta-induced proinflammatory genes at transcriptional level. The signals generated by CTS are sustained after its removal, and their persistence depends upon the length of CTS exposure. Furthermore, the sustained effects of mechanical signals are also reflected in their ability to induce aggrecan synthesis. These findings, once extrapolated to human chondrocytes, may provide insight in obtaining optimal sustained effects of physical therapies in the management of arthritic joints.

Fig. 1

Effect of CTS on iNOS induction in articular chondrocytes. Chondrocytes grown on Bioflex plates were exposed to CTS for 0, 4, 8 or 16 h, in the presence of IL-1β (inset A), or CTS followed by rest (CTS/rest) for 4/20, 8/16, 12/12, 16/8, 20/4 or 24/0 h, in the constant presence of IL-1β (A). The expression of iNOS mRNA was analyzed by real-time PCR (A, and inset A), and iNOS protein was assessed in immunostained chondrocytes by LSC (B). Analysis of iNOS mRNA expression in chondrocytes exposed to CTS/rest for 8/16, 8/28, or 8/ 40 h by real-time PCR (C), and total NO accumulation in the culture supernatants of chondrocytes exposed to CTS/rest for 8/16, 8/28, or 8/40 h (D). Data represent mean and s.e.m. of three separate experiments performed in triplicates. * indicates P < 0.05 as compared to IL-1β-treated cells.

Fig. 2

Effect of CTS on IL-1β-dependent COX-2 induction in articular chondrocytes. Chondrocytes grown on Bioflex plates, were exposed to CTS for 4 or 24 h, in the presence of IL-1β (inset A), or CTS/rest for 4/20, 8/16, 12/12, 16/8, 20/4 or 24/0 h, in the constant presence of IL-1β (A). The expression of COX-2 mRNA was analyzed by real-time PCR (A, and inset A), and COX-2 protein was assessed in immunostained chondrocytes by LSC (B). Microscopic representation of chondrocytes treated with CTS/rest for 0/24, 8/ 16, 16/8, or 24/0 h in the presence of IL-1β showing nuclear localization of COX-2. Chondrocytes were stained with anti-COX-2 antibodies to show the sustained inhibition of COX-2 synthesis in cells treated with CTS/rest for various time intervals (C). Data in (A and B) represent mean and s.e.m. of three separate experiments performed in triplicates. Micrographs in (C) represent one of three separate experiments. * indicates P < 0.05 as compared to IL-1β-treated cells.

Fig. 3

CTS-induced blocking of IL-1β-dependent induction of MMP-9 and MMP-13. Articular chondrocytes grown on flexible bottom plates were exposed to various durations of CTS or CTS/rest. Chondrocytes exposed to CTS for 4 and 24 h in the presence of IL-1β and analyzed for MMP-9 and MMP-13 by real-time PCR [inset Fig. 1(A)]. Chondrocytes exposed to CTS/rest for 4/20, 8/16, 12/12, 16/8, 20/4 or 24/0 h were analyzed by real-time PCR for MMP-9 and MMP-13 mRNA expression (A). Synthesis of MMP-9 was assessed by LSC evaluation of immunostained cells (B and C) and synthesis of MMP-13 was determined by densitometric analysis of Western Blots (B). Data represent mean and s.e.m. of three separate experiments. * indicates P < 0.05 as compared to IL-1β-treated cells. ❖ indicates P < 0.01 as compared to chondrocytes treated with CTS/rest for 8/16 h.

Fig. 4

Effect of CTS on IL-1β induced attenuation of aggrecan synthesis. Chondrocytes cultured on Bioflex plates were subjected to CTS or CTS/rest in the presence of IL-1β. Cells exposed to CTS/rest for 4/20, 8/16, 12/12, 16/8, 20/4, or 24/0, were analyzed for GAPDH and aggrecan mRNA expression by RT-PCR (inset A), and the intensity of each PCR product measured by densitometric analysis (A). RT-PCR analysis of chondrocytes exposed to CTS for 0, 4, or 24 h, in the presence of IL-1β (B). GAG synthesis in chondrocytes exposed to various periods of CTS/rest as shown in (A), was examined by Safranin-O staining, using dichromatic filter and Axioplan Software. The bars represent means and s.e.m. of Field Density Mean per 100 cells in five different areas of membrane, * represents P < 0.05 as compared to IL-1β-treated cells (C).