We have examined the role of cyclooxygenase 2 (COX-2) in a model of inflammaion in vivo. Carrageenan administration to the subcutaneous rat air pouch induces a rapid inflammatory response characterized by hih levels of prostaglandins (PGs) and leukotrienes in the fluid exudate. The time course of the induction of COX-2 mRNA and protein coincided with the production of PGs in the pouch tissue and cellular infiltrate. Carrageenan-induced COX-2 immunoreactivity was localized to macrophages obtained from the fluid exudate as well as to the inner surface layer of cells within the pouch lining. Dexamethasone inhibited both COX-2 expression and PG synthesis in the fluid exudate but failed to inhibit PG synthesis in the stomach. Furthermore, NS-398, a selective COX-2 inhibitor, and indomethacin, a nonselective COX-1/COX-2 inhibitor, blocked proinflammatory PG synthesis in the air pouch. In contrast, only indomethacin blocked gastric PG and, additionally, produced gastric lesions. These results suggest that inhibitors of COX-2 are potent antiinflammatory agents which do not produce the typical side effects (e.g., gastric ulcers) associated with the nonselective, COX-1-direc antiinfla tory drugs.Nonsteroidal antiinflammatory drugs (NSAIDs) are used to treat acute and chronic inflammatory disorders such as rheumatoid arthritis. The antiinflammatory mechanism of NSAIDs is due to a reduction ofprostaglandin (PG) synthesis by the direct inhibition of cyclooxygenase (COX; prostaglandin-endoperoxide synthase, EC 1.14.99.1) (1). Unfortunately, inhibition of PG production in organs such as stomach and kidney can result in gastric lesions, nephrotoxicity, and increased bleeding.COX exists in two forms. COX-1 is in most tissues and is involved in the physiological production of PGs. COX-2 is cytokine-inducible and is expressed in inflammatory cells (2-9). The identification of constitutive and inducible COX enzymes led to the hypothesis that COX-2 is primarily responsible for PGs produced in inflammation and COX-1 for PGs involved in normal homeostasis (4-6, 10, 11).The rat air pouch is a convenient model to study acute inflammation (12). It is formed by the subcutaneous injection of air over several days and is composed of a lining of cells that consists primarily of macrophages and fibroblasts. Injection of carrageenan into the fully formed air pouch produces an inflammatory granulomatous reaction characterized by a marked production of biochemical mediators in the fluid exudate, including PGs and leukotrienes, as well as a significant influx of polymorphonuclear leukocytes (PMNs) and macrophages (13). Using molecular and pharmacological reagents, we studied the role of COX-2 in this model of inflammation by specific examination of the induction of COX-2 mRNA and protein as well as the production of PGs in the pouch exudate. The results indicate that induction of COX-2 is responsible for the production of PGs at the site of inflammation, whereas the normal synthesis of PGs in the stomach appears to depend on constitutive...

1 The role of nitric oxide (NO) derived from constitutive and inducible nitric oxide synthase (cNOS and iNOS) and its relationship to oxygen-derived free radicals and prostaglandins (PG) was investigated in a carrageenan-induced model of acute hindpaw inflammation. 2 The intraplantar injection of carrageenan elicited an inflammatory response that was characterized by a time-dependent increase in paw oedema, neutrophil infiltration, and increased levels of nitrite/nitrate (NO2-/NO3-) and prostaglandin E2(PGE2) in the paw exudate. 3 Paw oedema was maximal by 6 h and remained elevated for 10 h following carrageenan administration. The non-selective cNOS/iNOS inhibitors, N0-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME) given intravenously (30-300 mg kg-') 1 h before or after carrageenan administration, inhibited paw oedema at all time points. 4 The selective iNOS inhibitors, N-iminoethyl-L-lysine (L-NIL) or aminoguanidine (AG), failed to inhibit carrageenan-induced paw oedema during the first 4 h following carrageenan administration, but inhibited paw oedema at subsequent time points (from 5-10 h). iNOS mRNA was detected between 3 to 10 h following carrageenan administration using ribonuclease protection assays. iNOS protein was first detected 6 h and was maximal 10 h following carrageenan administration as shown by Western blot analysis. Administration of the iNOS inhibitors 5 h after carrageenan (a time point where iNOS was expressed) inhibited paw oedema at all subsequent time points. Infiltrating neutrophils were not the source of iNOS since pretreatment with colchicine (2 mg kg-) suppressed neutrophil infiltration, but did not inhibit the iNOS mRNA expression or the elevated N02-/NO3-levels in the paw exudate. 5 Inhibition of paw oedema by the NOS inhibitors was associated with attenuation of both the N02-/NO3-and PGE2 levels in the paw exudate. These inhibitors also reduced the neutrophil infiltration at the site of inflammation. 6 Recombinant human Cu/Zn superoxide dismutase coupled to polyethyleneglycol (PEGrhSOD;12 x 103 u kg-'), administered intravenously either 30 min prior to or 1 h after carrageenan injection, inhibited paw oedema and neutrophil infiltration, but had no effect on NO2y/NO3-or PGE2 production in the paw exudate. The administration of catalase (40 x 103 u kg-'), given intraperitoneally 30 min before carrageenan administration, had no effect on paw oedema. Treatment with desferrioxamine (300 mg kg-'), given subcutaneously 1 h before carrageenan, inhibited paw oedema during the first 2 h after carrageenan administration, but not at later times. 7 These results suggest that the NO produced by cNOS is involved in the development of inflammation at early time points following carrageenan administration and that NO produced by iNOS is involved in the maintenance of the inflammatory response at later time points. The potential interactions of NO with superoxide anion and PG is discussed.

Objective To examine cyclooxygenase‐2 (COX‐2) enzyme expression, its regulation by interleukin‐1β (IL‐1β), and the role of prostaglandin E2 (PGE2) in proteoglycan degradation in human osteoarthritic (OA) cartilage. Methods Samples of human OA articular cartilage, meniscus, synovial membrane, and osteophytic fibrocartilage were obtained at knee arthroplasty and cultured ex vivo with or without IL‐1β and COX inhibitors. COX expression was evaluated by immunohistochemistry and Western blot analysis. The enzymatic activity of COX was measured by conversion of arachidonic acid to PGE2. Cartilage degradation was evaluated by measuring the accumulation of sulfated glycosaminoglycans in the medium. Results IL‐1β induced robust expression of COX‐2 and PGE2 in OA meniscus, synovial membrane, and osteophytic fibrocartilage explants, whereas low levels were produced in OA articular cartilage. IL‐1β also induced cartilage proteoglycan degradation in OA synovial membrane‐cartilage cocultures. Increased proteoglycan degradation corresponded to the induction of COX‐2 protein expression in, and PGE2 production from, the synovial membrane. Dexamethasone, neutralizing IL‐1β antibody, or the selective COX‐2 inhibitor, SC‐236, attenuated both the IL‐1β‐induced PGE2 production and cartilage proteoglycan degradation in these cocultures. The addition of PGE2 reversed the inhibition of proteoglycan degradation caused by SC‐236. Conclusion IL‐1β‐induced production of COX‐2 protein and PGE2 was low in OA articular cartilage compared with that in the other OA tissues examined. IL‐1β‐mediated degradation of cartilage proteoglycans in OA synovial membrane‐cartilage cocultures was blocked by the selective COX‐2 inhibitor, SC‐236, and the effect of SC‐236 was reversed by the addition of exogenous PGE2. Our data suggest that induction of synovial COX‐2‐produced PGE2 is one mechanism by which IL‐1β modulates cartilage proteoglycan degradation in OA.

This study shows that L-NIL reduces the progression of experimental OA. This effect could be related to a reduced level of chondrocyte apoptosis and is likely mediated by a decrease in the level of caspase 3 activity. A sparing effect of L-NIL on the increased level of Bcl-2 may also be a contributing factor.

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Objective. To evaluate the in vivo therapeutic efficacy of N-iminoethyl-L-lysine (L-NIL), a selective inhibitor of inducible nitric oxide synthase (iNOS), on the progression of lesions in an experimental osteoar-thritis (OA) dog model. The effect of L-NIL on metal-loprotease activity, levels of interleukin-1P (IL-lp), prostaglandin E, (PGE,), and nitritehitrate in synovial fluid was determined. Methods. The OA model was created by sectioning the anterior cruciate ligament of the right stifle joint of mongrel dogs by a stab wound. Dogs were separated into experimental groups: Group 1 was made up of uuoperated dogs that received no treatment, group 2 were operated dogs with no treatment, and group 3 were operated dogs that received oral L-NIL (10 mg/kg/twice daily) starting immediately after surgery. The OA dogs were killed at 10 weeks after surgery. Results. Experiments showed that dog OA cartilage explants in culture produced an increased amount of NO (nitrite). Immunohistochemical study demonstrated that this was due to an increased level of iNOS in chondrocytes. OA dogs treated with L-NIL showed a reduction in the incidence of osteophytes compared with the untreated OA dogs (58% versus 92%) as well as in their size (mean ?z SEM 1.92 * 0.58 mm versus 5.08 k Supported in part by grants from thc Arthritis Society and MoiisantoISearle USA. 0.66 mm). Macroscopically, L-NIL decreased the size of the cartilage lesions by-50% both on condyles and plateaus. 'The histologic severity of both the cartilage lesions and synovial inflammation was significantly decreased in the L-NIL-treated dogs. Treatment with L-NIL also significantly decreased both collagenase and general metalloprotease activity in the cartilage and the levels of IL-lp, PGE,, and nitritehitrate in synovial fluid. Conclusion. This study demonstrated the effectiveness of a selective inhibitor of iNOS, L-NIL, in attenuating the progression of experimental OA. The data suggest that L-NIL may act by reducing the activity of metalloproteases in cartilage and the production of IL-1p by synovium, both of which are known to play a major role in the pathophysiology of OA structural changes.

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