Research article

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

TT Chowdhury¹ , S Arghandawi¹ , J Brand² , OO Akanji¹ , DL Bader¹ , DM Salter² and DA Lee¹

¹  School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London, E1 4NS, UK

²  Queens Medical Research Institute, 47 Little France Cresent, Edinburgh University, EH16 4TJ. UK

author email corresponding author email

Arthritis Research & Therapy 2008, 10:R35doi:10.1186/ar2389

Published:	18 March 2008

Abstract

Background

Nitric 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.

Methods

Chondrocyte/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.

Results

IL-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.

Conclusion

These 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.