Objective: Chondrocytes have been shown to produce large amounts of nitric oxide (NO) when appropriately stimulated with proinflammatory cytokines or bacterial lipopolysaccharide (LPS). In view of recent observations underscoring profound phenotypic differences between superficial and deep articular chondrocytes, these studies investigated NO production, inducible NO synthase (iNOS) activity, and messenger RNA (mRNA) expression of superficial and deep cartilage explants and cells.
Methods: Superficial and deep bovine and human articular cartilage explants and isolated bovine chondrocytes were cultured in the presence of stimulating cytokines or LPS. NO was measured by the Griess reagent. Inducible NOS activity was quantitated by conversion of L-14 C-arginine to L-14C-citrulline. Inducible NOS mRNA expression was quantitated by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization.
Results: Superficial bovine cartilage explants stimulated with interleukin-1 alpha, LPS, or tumor necrosis factor alpha for 24 and 48 hours produced significantly more NO than did deep explants with all stimulants and at both times. Similar results were obtained with stimulated isolated superficial and deep cells. NO synthase activity, measured by the conversion of L-14C-arginine to L-14C-citrulline, paralleled NO production. Comparable results were obtained using explants from a normal human donor. Semiquantitation of iNOS mRNA by RT-PCR showed significantly larger amounts of PCR products in superficial cells and superficial explants. These results were confirmed by in situ hybridization of explants and isolated cells.
Conclusion: Increased NO production at the cartilage surface-synovial fluid interface may play an important role in the modulation of cartilage damage in inflammatory arthritis.