THE EXPRESSION AND REGULATION OF NITRIC-OXIDE SYNTHASE IN HUMAN OSTEOARTHRITIS-AFFECTED CHONDROCYTES - EVIDENCE FOR UP-REGULATED NEURONAL NITRIC-OXIDE SYNTHASE

Classically, osteoarthritis (OA) has been considered a noninflammatory disease. However, the detection of selected inflammatory mediators in osteoarthritic fluid, in the absence of significant inflammatory cell infiltrate, is increasingly appreciated. We sought to identify the in flammatory component in human OA-affected cartilage that may be involv ed in cartilage damage/destruction. Using Western blot analysis and an antibody to the conserved region of nitric oxide synthase (NOS), we h ave observed up-regulation of NOS, one of the ''key players'' of infla mmation, in chondrocytes of OA-affected patients. Remarkably, none of the cartilage samples examined from normal joints demonstrated detecta ble amounts of this NOS. Western blot analysis using the same alpha-NO S antibody indicated that this NOS from OA-affected cartilage (OA-NOS) was larger in size than (and distinct from) transfected human hepatoc yte or murine inducible NOS (iNOS) (150 versus 133 kD) and similar in size to neuronal constitutive NOS (ncNOS). Antibodies specific for iNO S showed binding to murine and human iNOS but not to OA-NOS, endotheli al constitutive NOS, or ncNOS. Antibodies specific for ncNOS bound to ncNOS and also to OA-NOS, but not to murine or human iNOS or endotheli al constitutive NOS. Incubation of OA cartilage in serum-free medium r esulted in spontaneous release, for up to 72 h, of substantial amounts of nitrite (up to similar to 80 mu M/100 mg wet tissue), which could be inhibited by at least 80% with various inhibitors of iNOS, includin g inhibitors of protein synthesis and transcription factor NF-kappa B, but which (unlike murine macrophage iNOS) was not sensitive to hydroc ortisone or TGF-beta. Exposure of OA-affected cartilage to interleukin 1 beta, tumor necrosis factor-alpha, and lipopolysaccharide resulted in similar to 20-50% augmentation of nitrite accumulation, which was a lso sensitive to cycloheximide and pyrrolidine dithiocarbamate. Hence, our data indicate that OA-NOS (based on immunoreactivity and molecula r weight) is similar to ncNOS and that it releases nitric oxide, which may contribute to the inflammation and pathogenesis of cartilage dest ruction in OA.