Dynamic compression counteracts IL-1β induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs

Abstract

BackgroundNitric oxide and prostaglandin E2 (PGE2play pivotal roles in both the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. These mediators are influenced by both IL-1β and mechanical loading, and involve alterations in the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes. To identify the specific interactions that are activated by both types of stimuli, we examined the effects of dynamic compression on levels of expression of iNOS and COX-2 and involvement of the p38 mitogen-activated protein kinase (MAPK) pathway.MethodsChondrocyte/agarose constructs were cultured under free-swelling conditions with or without IL-1β and/or SB203580 (inhibitor of p38 MAPK) for up to 48 hours. Using a fully characterized bioreactor system, constructs were subjected to dynamic compression for 6, 12 and 48 hours under similar treatments. The activation or inhibition of p38 MAPK by IL-1β and/or SB203580 was analyzed by western blotting. iNOS, COX-2, aggrecan and collagen type II signals were assessed utilizing real-time quantitative PCR coupled with molecular beacons. Release of nitrite and PGE2 was quantified using biochemical assays. Two-way analysis of variance and the post hoc Bonferroni-corrected t-test were used to examine data.ResultsIL-1β activated the phosphorylation of p38 MAPK and this effect was abolished by SB203580. IL-1β induced a transient increase in iNOS expression and stimulated the production of nitrite release. Stimulation by either dynamic compression or SB203580 in isolation reduced the IL-1β induced iNOS expression and nitrite production. However, co-stimulation with both dynamic compression and SB203580 inhibited the expression levels of iNOS and production of nitrite induced by the cytokine. IL-1β induced a transient increase in COX-2 expression and stimulated the cumulative production of PGE2 release. These effects were inhibited by dynamic compression or SB203580. Co-stimulation with both dynamic compression and SB203580 restored cytokine-induced inhibition of aggrecan expression. This is in contrast to collagen type II, in which we observed no response with the cytokine and/or SB203580.ConclusionThese data suggest that dynamic compression directly influences the expression levels of iNOS and COX-2. These molecules are current targets for pharmacological intervention, raising the possibility for integrated pharmacological and biophysical therapies for the treatment of cartilage joint disorders.

The Regional Sensitivity of Chondrocyte Gene Expression to Coactive Mechanical Load and Exogenous TNF-α Stimuli

“…Moderate levels of dynamic compression appear to inhibit some of the catabolic effects of interleukin-1 on articular chondrocytes [24,25]. However, proteoglycan (PG) loss in cartilage explants subjected to injurious compression and cultured in the presence of TNF-a was greater than that produced by either injurious compression or TNF-a treatment alone [26].…”

“…Importantly, the two-week time course of the study by Ferretti et al [32] was much longer than the time course of this study. Further, low levels of in vitro dynamic compression seem to inhibit the catabolic effects of cytokines on articular chondrocytes [24,25,33], but a loading threshold may exist above which the protective effects of mechanical load are not observed [33]. Consistent with the findings of Li et al [33], dynamic unconfined compression was observed to upregulate CII and aggrecan expression relative to unloaded samples treated with TNF-a and TNF-a treatment was observed to upregulate ADAM-TS expression even in the presence of dynamic compression.…”

“…The interactive effects of mechanical stimuli and cytokines on articular cartilage have been investigated in in vivo [32] and in vitro studies [24,25,33] and the interaction may be dependent on the duration and magnitude of mechanical loading. Continuous passive motion, a low-magnitude load stimulus, attenuated cartilage degradation relative to immobilization in an in vivo model of ag-induced arthritis in rabbits [32].…”

“…In vitro studies [24,25] have demonstrated that the specific effects of certain cytokines may be modulated through mechanical loading. Moderate levels of dynamic compression appear to inhibit some of the catabolic effects of interleukin-1 on articular chondrocytes [24,25].…”

Regulation of Chondrocytic Gene Expression by Biomechanical Signals

“…The compression also prevented downregulation of ACAN in the presence of inflammation. 3,6 Similarly, compressive forces inhibited IL-1β-inducible nitric oxide synthase (INOS/NOS2A) and cyclooxygenase 2 (COX2)/prostaglandin G/H synthase 2 (PTGS2) expression 17,18 and upregulated PG synthesis and cell division in the presence or absence of IL-1β (Fig. 2).…”