by Inhibiting Inducible Nitric Oxide Synthase Antiinflammatory Actions on Chondrocytes Cyclic Tensile Stress Exerts

Continuous passive motion manifests therapeutic effects on inﬂamed articular joints by an as-yet-unknown mechanism. Here, we show that application of cyclic tensile stress (CTS) in vitro abrogates the catabolic effects of IL-1 b on chondrocytes. The effects of CTS are mediated by down-regulation of IL-1 b -dependent inducible NO production, and are directly attributed to the inhibition of inducible NO synthase (iNOS) mRNA expression and protein synthesis. The inhibition of iNOS induction by CTS is paralleled by abrogation of IL-1 b -induced down-regulation of proteoglycan synthesis. Furthermore, CTS inhibits iNOS expression and up-regulates proteoglycan synthesis at concentrations of IL-1 b frequently observed in inﬂamed arthritic joints, suggesting that the actions of CTS may be clinically relevant in suppressing the sustained effects of pathological levels of IL-1 b in vivo. These results are the ﬁrst to demonstrate that mechanisms of the intracellular actions of CTS in IL-1 b -activated chondrocytes are mediated through inhibition of a key molecule in the signal transduction pathway that leads to iNOS expression. The Journal of Immunology, 1999, 163: 2187–2192. IL-1 synovial ﬂuids of pliﬁed with the iNOS primers to obtain MIMIC DNA. The gene products of target and competitor sequences were thus 243 and 438 bp, respectively. The target cDNA derived from 0.033 m g of cellular RNA was then ampliﬁed in the presence of iNOS primers and 1 m l of MIMIC DNA (10 4 attomoles or its 10-fold serial dilutions). The lanes exhibiting similar concentrations of gene products in target and MIMIC DNA were reanalyzed to derive exact concentrations of target cDNA using 2-fold dilutions of the MIMIC DNA. The bands with equimolar concentration of the gene prod- ucts were estimated by densitometric analysis of ethidium bromide-stained DNA in each lane. The results were calculated and expressed as the mean number of iNOS mRNA molecules synthesized per m g RNA. Nonidet P-40. Lysates were gently transferred to tubes, centrifuged at 13,000 rpm for 30 sec to pellet nuclei, and the supernatant proteins (10 m g/lane)were subjected to SDS-10% PAGE. The proteins were then electrophoretically transferred to Polyscreen polyvinylidene diﬂuoride transfer membrane (Dupont, Wilmington, DE), and the resulting blot was blocked with 5% BSA and 5% nonfat milk in PBS. The blot was then reacted with rabbit anti-iNOS-2 (Transduction Laboratories, Lexington, KY) Abs at 4°C over-night, and washed with PBS containing 0.2% Tween-20. The binding of primary Abs was detected by HRP-conjugated goat anti-rabbit IgG, followed by chemiluminescent substrate Luminol (New England Nuclear, Boston, MA) for 60 s. The blots were exposed to a Reﬂection NEF-496 ﬁlm (Dupont) for 30–60 s to visualize iNOS. For immunoperoxidase staining, chondrocytes (10 5 /well) were trypsinized from Flexercell wells, cytocentrifuged onto microscope slides, and ﬁxed for 30 min in ice-cold methanol containing 0.3% hydrogen per-oxide to inactivate endogenous peroxidases (29). iNOS was detected by the use of rabbit anti-NOS-2 as primary Abs, and HRP-conjugated goat anti-rabbit IgG as secondary Abs. The chromogen used for HRP detection was diaminobenzidine. The relative intensity of immunoperoxidase staining for iNOS was determined by histomorphometric densitometry using captured images and Optimus software (Media Cybernetics, Silver Spring, MD).