Cartilage specimens from osteoarthritis (OA)-affected patients spontaneously released PGE 2 at 48 h in ex vivo culture at levels at least 50-fold higher than in normal cartilage and 18-fold higher than in normal cartilage ϩ cytokines ϩ endotoxin. The superinduction of PGE 2 production coincides with the upregulation of cyclooxygenase-2 (COX-2) in OA-affected cartilage. Production of both nitric oxide (NO) and PGE 2 by OA cartilage explants is regulated at the level of transcription and translation. Dexamethasone inhibited only the spontaneously released PGE 2 production, and not NO, in OA-affected cartilage. The NO synthase inhibitor H N G -monomethyl-L -arginine monoacetate inhibited OA cartilage NO production by Ͼ 90%, but augmented significantly (twofold) the spontaneous production of PGE 2 in the same explants. Similarly, addition of exogenous NO donors to OA cartilage significantly inhibited PGE 2 production. Cytokine ϩ endotoxin stimulation of OA explants increased PGE 2 production above the spontaneous release. Addition of L -NMMA further augmented cytokine-induced PGE 2 production by at least fourfold. Inhibition of PGE 2 by COX-2 inhibitors (dexamethasone or indomethacin) or addition of exogenous PGE 2 did not significantly affect the spontaneous NO production. These data indicate that human OA-affected cartilage in ex vivo conditions shows ( a ) superinduction of PGE 2 due to upregulation of COX-2, and ( b ) spontaneous release of NO that acts as an autacoid to attenuate the production of the COX-2 products such as PGE 2 . These studies, together with others, also suggest that PGE 2 may be differentially regulated in normal and OA-affected chondrocytes. ( J. Clin. Invest. 1997. 99:1231-1237.)

Tetracyclines have recently been shown to have ''chondroprotective'' effects in inf lammatory arthritides in animal models. Since nitric oxide (NO) is spontaneously released from human cartilage affected by osteoarthritis (OA) or rheumatoid arthritis in quantities sufficient to cause cartilage damage, we evaluated the effect of tetracyclines on the expression and function of human OA-affected nitric oxide synthase (OA-NOS) and rodent inducible NOS (iNOS). Among the tetracycline group of compounds, doxycycline > minocycline blocked and reversed both spontaneous and interleukin 1␤-induced OA-NOS activity in ex vivo conditions. Similarly, minocycline > doxycycline inhibited both lipopolysaccharide-and interferon-␥-stimulated iNOS in RAW 264.7 cells in vitro, as assessed by nitrite accumulation. Although both these enzyme isoforms could be inhibited by doxycycline and minocycline, their susceptibility to each of these drugs was distinct. Unlike acetylating agents or competitive inhibitors of L-arginine that directly inhibit the specific activity of NOS, doxycycline or minocycline has no significant effect on the specific activity of iNOS in cell-free extracts. The mechanism of action of these drugs on murine iNOS expression was found to be, at least in part, at the level of RNA expression and translation of the enzyme, which would account for the decreased iNOS protein and activity of the enzyme. Tetracyclines had no significant effect on the levels of mRNA for ␤-actin and glyceraldehyde-3-phosphate dehydrogenase nor on levels of protein of ␤-actin and cyclooxygenase 2 expression. These studies indicate that a novel mechanism of action of tetracyclines is to inhibit the expression of NOS. Since the overproduction of NO has been implicated in the pathogenesis of arthritis, as well as other inf lammatory diseases, these observations suggest that tetracyclines should be evaluated as potential therapeutic modulators of NO for various pathological conditions.

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Nitric oxide synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation in rheumatic and autoimmune diseases. We report that exposure of lipopolysaccharide-stimulated murine macrophages to therapeutic concentrations of aspirin (IC50 = 3 mM) and hydrocortisone (IC50 = 5 ,uM) inhibited the expression of iNOS and production of nitrite. In contrast, sodium salicylate (1-3 mM), indomethacin (5-20 ,uM), and acetaminophen (60-120 ,uM) had no significant effect on the production of nitrite at pharmacological concentrations. At suprapharmacological concentrations, sodium salicylate (IC50 = 20 mM) significantly inhibited nitrite production.Immunoblot analysis of iNOS expression in the presence of aspirin showed inhibition of iNOS expression (IC5s = 3 mM). Sodium salicylate variably inhibited iNOS expression (0-35%), whereas indomethacin had no effect. Furthermore, there was no significant effect of these nonsteroidal antiinflammatory drugs on iNOS mRNA expression at pharmacological concentrations. The effect of aspirin was not due to inhibition of cyclooxygenase 2 because both aspirin and indomethacin inhibited prostaglandin E2 synthesis by >75%. Aspirin and N-acetylimidazole (an effective acetylating agent), but not sodium salicylate or indomethacin, also directly interfered with the catalytic activity of iNOS in cell-free extracts. These studies indicate that the inhibition of iNOS expression and function represents another mechanism of action for aspirin, if not for all aspirin-like drugs. The effects are exerted at the level of translational/posttranslational modification and directly on the catalytic activity of iNOS.Nitric oxide (NO), first identified as an endothelium-derived relaxation factor (1), is now recognized to be an intra-and extracellular mediator of cell function (2-5). NO produced by the constitutive isoform of nitric oxide synthase (NOS) is a key regulator of homeostasis, whereas the generation of NO by inducible NOS (iNOS) plays an important role in inflammation, host-defense responses, and tissue repair (2-4). NO formation is increased during inflammation (rheumatoid arthritis, and ulcerative colitis, Crohn disease), and several classic inflammatory symptoms (erythema and vascular leakiness) are reversed by NOS inhibitors (2-4). Vane and coworkers (6) have implicated NO as an important mediator of inflammation in animal models. Furthermore, because iNOS is up-regulated by endotoxin, interleukin 1, tumor necrosis factor, and interferon y, the increased synthesis of NO has been implicated in autoimmune diseases, allograft rejection, graft-versus-host disease, and systemic response to sepsis. Recent studies by Salvemini et al. (7) have shown that NO modulates the activity of prostaglandin endoperoxide H synthase 2 [cyclooxygenase 2 (COX-2)] in a concentrationdependent manner, through a mechanism independent of cGMP.Although nonsteroidal antiinflammatory drugs (NSAIDs) clearly inhibit the synthesis and release of prostaglandins (8, 9), these actions ar...