Regulation of Nitric Oxide Production by Salicylates and Tenidap in Human OA-Affected Cartilage, Rat Chondrosarcomas and Bovine Chondrocytes by Mukundan G

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provided by Elsevier - Publisher Connector Osteoarthritis and Cartilage (1998) 6, 269–277 7 1998 Osteoarthritis Research Society 1063–4584/98/040269 + 09 $12.00/0 Article No. oc980120

Regulation of nitric oxide production by salicylates and tenidap in human OA-affected cartilage, rat chondrosarcomas and bovine chondrocytes By Mukundan G. Attur*, Rajesh Patel*, Paul E. DiCesare†, German C. Steiner‡, Steven B. Abramson and Ashok R. Amin*‡§ *Department of Rheumatology, Hospital for Joint Diseases, New York; †Department of Orthopaedics, Hospital for Joint Diseases, New York; ‡Department of Pathology, Kaplan Cancer Center, New York University Medical Center, New York; §Department of Medicine, New York University Medical Center, New York, U.S.A.

Summary Objective: To examine the effects of non-steroidal anti-inflammatory drugs (NSAIDS) on nitric oxide (NO) and prostaglandin E2 (PGE2) production in chondrocytes from three different species. Methods: We have estimated NO production by Griess method, and PGE2 by RIA from the supernatants of articular cartilage obtained from osteoarthritis joints (OA-affected cartilage), rat chondrosarcomas (in ex vivo conditions) and bovine chondrocytes (stimulated with cytokines + endotoxin in in vitro conditions) in the presence or absence of aspirin, indomethacin, sodium salicylate, tenidap and glucocorticoids. Results: NO, which was spontaneously released in ex vivo conditions by OA-affected cartilage and rat chondrosarcomas (maintained in vivo), was susceptible to inhibition by pharmacologically relevant concentrations of aspirin, sodium salicylate and tenidap, but not to concentrations of indomethacin or glucocorticoids that significantly inhibited PGE2 production under the same conditions. Similarly, the production of NO by bovine chondrocytes grown in monolayer cultures that had been stimulated with cytokines + endotoxins (in vitro) to release both NO and PGE2 (at 48–72 h post stimulation), were inhibited by aspirin, sodium salicylate and tenidap, but not by indomethacin or glucocorticoids at concentrations sufficient to inhibit PGE2 production. Inhibition of NO in the cytokines + endotoxin stimulated bovine chondrocytes (like the human OA-affected cartilage) augmented PGE2 production. Conclusion: These experiments demonstrate that NO production by chondrocytes across species show a similar profile of susceptibility to inhibition by selected anti-inflammatory drugs. The insensitivity of NO production to glucocorticoids is an important characteristic of these cells that merits further investigation. Key words: Non-steroidal anti-inflammatory drugs, Osteoarthritis, Nitric oxide, Prostaglandin.

Introduction human studies. Mean serum nitrite concentrations in individuals affected by rheumatoid arthritis Inducible nitric oxide synthase (iNOS) plays an (RA) and osteoarthritis (OA) are significantly important role in inflammation, host-defense higher than those observed in normal controls [5]. responses, and tissue repair [1]. Nitric oxide (NO) In both disease groups, nitrite concentrations were formation is increased during inflammation (e.g., significantly higher in synovial fluid than serum, rheumatoid arthritis, ulcerative colitis or Crohn’s implying synthesis of NO within diarthrodial disease) and several classic inflammatory symp- joint(s) where both synoviocytes and chondrocytes toms such as erythema and vascular leakiness [2]. have been implicated as sites of increased NO The role of NO has been well demonstrated in production [6, 7]. Previous studies have also shown various animal models of arthritis [3, 4]. The that OA/RA-affected cartilage in ex vivo conditions increased production of NO characteristic of the release NO in sufficient quantities to cause rodent models of arthritis has been observed in cartilage dysfunction [6, 7]. Recent studies have Received 15 August 1997; accepted 6 March 1998. suggested that superficial and deep human and Supported by a grant from Pfizer Inc., U.S. Pharmaceuticals bovine articular chondrocytes show differential Group, New York, U.S.A. production of NO [8, 9]. Excessive concentrations Address correspondence to: Ashok R. Amin, Ph.D., Hospital for Joint Diseases, 301 East 17th Street, Room 1600, New York, NY of nitric oxide exert profound effects on chondro- 10003, U.S.A. Tel: 212-598-6537, Fax: 212-598-7604. cyte functions, including: down regulation of

269 270 Attur et al.: Effect of NSAIDS on NO and PGE2 in chondrocytes collagen synthesis [10], inhibition of actin polym- with the diagnosis of advanced OA (age 50–70 erization [11], down regulation of interleukin years) who were undergoing knee replacement IL-1RA expression [12] and induction of apoptosis surgery. [13]. Furthermore, two groups have independently presented evidence to suggest that NO induces the procurement of bovine chondrocytes synthesis of matrix metalloproteases by monolayer cultures of articular chondrocytes [14, 15]. These The bovine cartilage was obtained from young manifestations induced by NO, collectively may be calfs. Isolation of bovine chondrocytes from hoofs a possible mechanism of pathological neovas- was carried out by standard methods [18, 19] with culization in arthritis. minor modifications. Briefly, normal bovine carti- Although non-steroidal anti-inflammatory drugs lage was washed in RPMI-1640 and cut into small (NSAIDS) clearly inhibit the synthesis and release pieces and digested with enzymes. Cartilage pieces of prostaglandins, these actions are not sufficient were incubated with trypsin (0.25%) in RPMI-1640 to explain all the anti-inflammatory effects of (Gibco BRL, Gaithersburg, MD, U.S.A.) for 30 min NSAIDS. For example, NSAIDS also inhibit at 37°C before they were washed and re-incubated activation of neutrophils, which release inflamma- in hyaluronidase (0.2%) and collagenase (0.2%), tory mediators other than prostaglandins [16]. dissolved in RPMI-1640 containing 5% fetal bovine We have recently observed that several anti- serum (FBS) for 16 h at 37°C with continuous inflammatory drugs, (namely salicylates, glucocor- agitation (100 rpm). The cells were passed through ticoids and tetracyclines) inhibit iNOS expression 75 mm nylon mesh and washed twice with PBS to in murine macrophages stimulated with lipo- remove cell debris. The released cells were polysaccharides (LPS) [17]. In the present study, suspended in RPMI 1640 + 10% FBS + antibiotics we investigated the capacity of NSAIDS to inhibit and plated in a 24-well plate (Becton Dickinson, 5 spontaneous release of NO and prostaglandin E2 Lincoln Park, NJ, U.S.A.) at a density of 5 × 10 2 (PGE2) by OA-affected cartilage and rat chon- cells/2.0 cm . After 48 h, the medium was changed drosarcoma cells (in ex vivo conditions). We also and the cells were preincubated in fresh media investigated the action of NSAIDS on in vitro with various modulators (NSAIDS) for 2 h before cytokine [IL-1b and tumor necrosis factor stimulating them with IL-1b (1 ng/ml) + TNFa (TNFa)] + endotoxin stimulated bovine chondro- (100 U/ml) and LPS 100 mg/ml) and analyzed for NO cytes producing NO and PGE2. We report that and PGE2 levels at 48–72 h of post stimulation. aspirin, sodium salicylate and tenidap, but not Ethanol was used as a carrier for the NSAIDS and indomethacin, inhibit NO production in human carrier concentration was maintained ( Q 0.01% OA-affected cartilage, chondrosarcomas, and v/v) in the cultures. Equivalent amount of ethanol bovine chondrocytes. The failure of glucocorticoid was also added to the control cultures. Culture to inhibit NO production in human chondrocytes, media were assayed for NO and PGE2 at different rat chondrosarcomas and bovine chondrocytes is time intervals. distinct from the previous report of its effects on murine macrophages. isolation and maintenance of rat chondrosarcoma cells Methods A Swarm rat chondrosarcoma cell line was reagents and cell lines maintained in Sprague–Dawley rats as previously described [20]. Briefly, 100 g Sprague–Dawley male RPMI-1640 and Ham’s F-12 were purchased from rats were subcutaneously implanted with the Mediatech Inc. (Hernden, VA, U.S.A.). The Swarm rat chondrosarcoma. The Swarm rat endotoxin level in this media was Q 0.01 ng/ml. chondrosarcoma is a well-differentiated malignant LPS, NSAIDS and steroids were obtained from tumor that resembles a well-differentiated human Sigma (St. Louis, MO, U.S.A.) Cytokines were chondrosarcoma [21]. After 3 weeks, the animals purchased from Fischer Scientific (Pittsburgh, PA, were sacrificed by CO2 asphyxiation and tumor U.S.A.). Tenidap was kindly provided by Pfizer, harvested using sterile technique. The solid tumor Inc. (Groton, CT, U.S.A.). was minced with scissors, centrifuged, and digested with 0.5 mg/ml hyaluronidase (Sigma, MO, U.S.A.) for 10 min followed by 0.2% (w/v) collagen- procurement of human cartilage ase (Worthington Biochem. Co., NJ, U.S.A.) for 2 h Cartilage slices were taken from the knees in Hams-F12 media (Life Technologies Inc., Grand (tibial plateau and femoral condyle) of patients Island, NY, U.S.A.), 10 mm Hepes, pH 7.4, Osteoarthritis and Cartilage Vol. 6 No. 4 271 containing 50 mg/ml gentamicin in a Spinner Results culture flask at 37°C. The cells were filtered We have recently reported that aspirin (150 m nylon mesh) to remove remaining large m and sodium salicylate, but not indomethacin cellular debris. could inhibit NO production in murine macro- Cells were washed and resuspended in Hams-F12 phages in a PGE /COX-2 independent fashion [17]. media, 50 g/ml gentamicin, 10 mm Hepes, pH 7.4, 2 m We have now extended those studies to three containing 1% fetal calf serum (FCS) at a final chondrocyte systems, each of different species. concentration of 5 × 105/ml. The cells were then These include human OA-affected cartilage, transferred to a 24-well cell culture plates (5 × 105 Swarm rat chondrosarcoma cells and bovine cells/well) and allowed to attach for 24 h. After chondrocytes. 24 h, the medium was changed and NSAIDS were In an effort to elucidate the effect of NSAIDS on added. Ethanol was used as a carrier. NOS expression and function in cartilage and chondrocytes, we have selected four known human oa-explant assay NSAIDs: an acetylated salicylate (aspirin, an effective inhibitor of COX-2 at the concentrations Total knee articular cartilage from tibial studied); a non-acetylated salicylate (sodium plateau and femoral condyle were harvested from salicylate, a less effective inhibitor of COX-2) OA-affected patients. The cartilage was mixed and indomethacin (a potent inhibitor of COX-2) and cut into 3-mm discs and four to six discs were tenidap (an anti-inflammatory drug that inhibits placed in triplicate or quadruplicate, in a 24-well COX-2 as well as IL-1 production) [24]. We tested plate in 2.0 ml Ham’s F-12 medium supplemented b the hypothesis that NSAIDS, which inhibited NO with 0.2% endotoxin free human albumin, 10 mm production in murine macrophages also modulates Hepes at pH 7.4 and antibiotics in the presence of NOS in cartilage and chondrocytes in a COX-2 various modulators, as previously described [6, 22]. independent manner across species. Salicylates, The NSAIDS, tenidap, and glucocorticoids were indomethacin and tenidap, which reach thera- used at pharmacologically relevant concentrations peutic concentrations in plasma of 1–3 mm, in ethanol [ Q 0.01% (v/v) final concentration] as 5–20 m, and 30 m, respectively [25] were tested for carrier. Equivalent amount of ethanol was added m m their capacity to inhibit NO production at the in all cultures. clinically relevant concentrations. For the present study we selected three model

determination of pge2 and nitrite systems for the study of chondrocytes: (a) human OA-affected cartilage, (b) rat chondrosarcoma cells PGE was determined in the culture supernatant 2 and (c) bovine chondrocytes. In the OA-affected using a radio-immunoassay (RIA) kit (Sigma, MO, cartilage and rat chondrosarcoma cells, the NO U.S.A.) according to the manufacturer’s instruc- (induced in vivo) was spontaneously released in ex tions. The detection limit was 1.0 pg/ml. NO vivo conditions [6, 26]. The NOS isoforms expressed production was measured by estimating the stable in OA-affected cartilage and rat chondrosarcoma NO metabolite, nitrite, in conditioned medium, by cells are different in view of the 150 kDa OA-NOS a modiﬁed Griess reaction [23]. The values were in OA-affected cartilage [6] and 133 kDa iNOS in expressed as micromolar nitrite or ng/ml PGE 2 the rat chondrosarcoma cells [26]. The third model released per 100 mg wet weight of cartilage and system was bovine chondrocytes, which when 5 × 105 of chondrocytes. grown in monolayer culture, produces NO (and express a 133 kDa iNOS) upon in vitro stimulation statistical analysis with cytokines + endotoxin (Fig. 1). The data represents a minimum of one of the two experiments performed from separate OA-affected effect of nsaids on no production in cartilage samples. All experiments using cartilage oa-affected cartilage under ex vivo slices, rat chondrosarcoma cells or bovine chon- conditions drocytes were performed in triplicate or quadruplicate for each parameter studied. Data are Human OA-affected cartilage explants in ex vivo expressed as mean 2 s.d. and statistical analysis conditions were supplemented with aspirin, was performed using GraphPAD Software (v 1.14). sodium salicylate and indomethacin as shown in The t test or non parametric (Mann–Whitney or Tables I and II. The spontaneous release of NO was Wilcoxon) test were performed for all the monitored at different time intervals. As expected, experiments. time dependent spontaneous release of NO in 272 Attur et al.: Effect of NSAIDS on NO and PGE2 in chondrocytes

consistently and signiﬁcantly inhibited the spontaneous release of NO in OA-affected cartilage, but showed variable effects (0–30% inhibition at similar concentrations) in murine macrophages [27]. OA-affected cartilage spontaneously releases

signiﬁcant quantities of PGE2 via upregulation of COX-2 not seen in normal cartilage [22]. We therefore tested the ability of these NSAIDS to

inhibit spontaneously released PGE2 in OA-af- Fig. 1. Western blot analysis of iNOS from murine fected cartilage in ex vivo assays. As expected, macrophages and bovine chondrocytes. Murine macro- aspirin (1–3 mm), sodium salicylate (1–3 mm) and phages (RAW 264.7 cells) were stimulated with 1 mg/ml indomethacin (20 mm) significantly inhibited PGE2 of LPS for 24 h and cells were harvested. Bovine production (by q 90%) (Table III). chondrocytes were seeded for 48 h. After 48 h the The effect of dexamethasone was examined on adherent cells were stimulated with IL-1b (1 ng/ml) + TNFa (100 U/ml) and LPS (100 mg/ml) for 48 h before the NO production in OA-affected cartilage explants. cells were harvested. The cell free extracts were One micromolar of dexamethasone, which signifi- prepared as previously described [25]. One hundred cantly inhibited PGE2 production, had no signifi- micrograms of protein was loaded on the sodium dodecyl cant effect on the spontaneous release of NO in the sulfate—polyacrylamide gel (SDS-PAGE) and western same explant (Table III). blotted using an anti-iNOS monoclonal antibody from Transduction Labs (Lexington, KY, U.S.A.) which cross Tenidap has recently been reported to inhibit reacts with bovine 133 kDa iNOS. synthesis of various cytokines which include IL-1b and IL-6 [24]. In three separate experiments tenidap (at 30 mm) also significantly inhibited the spontaneous release of NO from OA-affected cartilage. Tenidap also significantly inhibited the IL-1b mediated NO production in OA-affected OA-affected cartilage could be inhibited with cartilage (Table IV). pharmacological relevant concentrations of aspirin, sodium salicylate, but not indomethacin. The effect of nsaids on inos expressed in rat NOS competitive inhibitor (L-NMA) blocked NO chondrosarcoma in ex vivo conditions release in both experiments. These results (with respect to the effects of aspirin and indomethacin) The rat chondrosarcoma when maintained in were similar to those previously seen with murine rats (in vivo) shows upregulation of iNOS macrophages stimulated with LPS in the presence expression and spontaneously releases NO in of NSAIDS [27]. Sodium salicylate (at 2-3 mm) ex vivo conditions in the absence of any stimulus

Table I Effect of aspirin and sodium salicylate on spontaneous release of nitric oxide from OA-affected cartilage in ex vivo conditions mm nitrite/100 mg Cartilage

Conditions 24 h 48 h 72 h Control 4.7 2 1.5 10.1 2 1.6 12.9 2 1.2 + L-NMA (500 mm) 0.9 2 0.5b 0.7 2 0.3a 3.8 2 1.3a + Aspirin (1 mm) 2.6 2 0.1c 2.6 2 0.2a 8.5 2 2.0c + Aspirin (2mm) 2.7 2 1.1c 4.2 2 0.4a 7.5 2 2.6b + Aspirin (3 mm) 2.6 2 1.5c 3.4 2 1.5a 5.2 2 0.9a + Aspirin (5 mm) 1.8 2 0.0b 2.6 2 0.2a 3.5 2 0.4a + Sodium salicylate (2 mm) 6.8 2 4.9d 7.9 2 4.0c 10.3 2 4.9d + Sodium salicylate (3 mm) 3.4 2 0.3c 6.4 2 0.4c 8.2 2 1.9b OA-affected cartilage from patients undergoing knee replacement surgery was incubated in medium with various NSAIDS and NOS inhibitor. Nitrite levels were estimated as described in Methods. Data represent mean 2 .. value. The P-values described are compared with control unstimulated OA-cartilage. The data represents one of the three similar experiments. (P-values a R 0.0001, b R 0.001, c R 0.01, d R 0.1). Osteoarthritis and Cartilage Vol. 6 No. 4 273

Table II Effect of NSAIDS on spontaneous release of nitric oxide by OA-affected cartilage in ex vivo conditions mm nitrite/100 mg Cartilage

Conditions Experiment I Experiment II Control 6.2 2 2.6 28.3 2 12.7 + L-NMA (500 mm) ND 3.9 2 2.7a + Aspirin (2 mm) 3.3 2 0.7b 7.1 2 4.2b + Aspirin (3 mm) 1.6 2 0.5a ND + Sodium salicylate (2 mm) 2.5 2 0.5b 21.8 2 17.2c + Sodium salicylate (3 mm) 2.6 2 0.4b ND + Indomethacin (5 mm) 5.2 2 1.3c ND + Indomethacin (20 mm) ND 28.6 2 16.7c OA-affected cartilage was taken from patients undergoing knee replacement surgery and incubated in medium for 48 h. Experiments I and II represent OA-affected cartilage obtained from two separate patients. Nitrite levels were estimated as described in Methods. Data represent mean 2 .. value. The P-values described are compared with control (unstimulated) OA-affected cartilage. ND = not done. The data represents two similar experiments. (P-values a R 0.001, b R 0.01, c R 0.5).

[26] (Table V). L-NMA significantly inhibits the endotoxin. Bovine chondrocytes and murine spontaneous release of NO in ex vivo conditions. macrophages expressed a 133 kDa iNOS as shown Rat chondrosarcoma cells were exposed to in Fig. 1. Bovine chondrocytes grown in monolayer various NSAIDS in ex vivo conditions to examine for 48 h were stimulated with IL-1b + TNFa + LPS their effects on NO production. Aspirin and in the presence and absence of NSAIDS, and the sodium salicylate, but not indomethacin, signifi- levels of NO and PGE2 accumulated in the medium cantly inhibited (in a time- and dose-dependent were examined at different time intervals. Aspirin, manner) the release of NO from the rat chondro- sodium salicylate, and tenidap, but not in- sarcoma cells cultured in ex vivo conditions. In domethacin or hydrocortisone, significantly inhib- contrast, hydrocortisone (at 1 mm) did not inhibit ited the accumulation of NO in the medium (Table the NO production in chondrosarcoma cells. VI). The concentration of indomethacin and

hydrocortisone was sufﬁcient to inhibit PGE2 production in these cells at 72 h. effect of nsaids on bovine chondrocytes stimulated with cytokine + endotoxins in in vitro conditions Discussion We examined the expression of iNOS in Our experiments indicate that selected anti- bovine chondrocytes stimulated with cytokine + inﬂammatory drugs, including the salicylates and

Table III Regulation of nitric oxide and PGE2 in OA-affected cartilage in the presence of NSAIDS per 100 mg Cartilage

Conditions mm Nitrite PGE2 ng/ml Control 6.2 2 2.6 58.2 2 12.0 + Aspirin (2 mm) 3.3 2 0.7b 0.2 2 0.1a + Aspirin (3 mm) 1.6 2 0.5c 1.6 2 0.1a + Sodium Salicylate (2 mm) 2.5 2 0.5c 5.1 + 0.6a + Sodium Salicylate (3 mm) 2.6 2 0.4c 4.6 2 1.1a + Indomethacin (5 mm) 5.2 2 1.3d 1.3 2 0.1a + Dexamethasone (0.1 mm) 7.1 2 1.4d 2.9 2 0.6a + Dexamethasone (1.0 mm) 6.5 2 0.9d 4.1 2 0.2a OA-affected cartilage from patients undergoing knee replacement surgery were incubated in the medium with various NSAIDS. The nitrite and PGE2 levels were estimated from the same experiment as described in Methods. Data represents mean 2 .. value. The P-values described are compared with control unstimulated OA-affected. The data represents one of the two similar experiments. (P-values a R 0.0001, b R 0.001, c R 0.01, d R 0.5). 274 Attur et al.: Effect of NSAIDS on NO and PGE2 in chondrocytes

Table IV Effect of tenidap on nitric oxide production in OA-affected cartilage in ex vivo condition mm nitrite/100 mg cartilage

Experiment I Experiment II Experiment III

Conditions 24 h 48 h 72 h 48 h 72 h 24 h Control 1.7 2 0.3 4.2 2 0.6 4.7 2 0.8 2.9 2 1.2 3.1 2 1.4 2.1 2 0.3 + Tenidap (30 mm) 1.2 2 0.2b 2.1 2 1.2b 2.3 2 1.4b Q 0.1a Q 0.1a 1.4 2 0.2d + IL-1 (5 ng/ml) 6.1 2 1.5a 13.4 2 2.5a 16.4 2 3.9a 6.4 2 1.6a 9.4 2 1.4a 3.6 2 0.8b + IL-1 + Tenidap (30 mm)* 4.6 2 0.5c 9.2 2 0.7b 10.9 2 0.5b 2.8 2 0.8a 4.6 2 1.4a ND + Aspirin (2 mm) ND ND ND 0.2 2 0.3a 0.1 2 0.2a ND + IL-1 + Aspirin (2 mm)* ND ND ND 2.3 2 0.2a 2.4 2 0.3a ND OA-affected cartilage was taken from patients undergoing knee replacement surgery and incubated in medium for 48 h. Nitrite levels were estimated as described in Methods. Experiment I, II, III represent OA-affected cartilage obtained from different patients. Data represent mean 2 .. value. The P-value described a R 0.001, b R 0.01, c R 0.05, d R 0.1) are compared with control (unstimulated) OA-affected cartilage. The * (IL-1b + tenidap/Aspirin) are compared with the IL-1b stimulated OA-affected cartilage. ND: not done.

tenidap, inhibit NO production by chondrocytes of the salicylates, exert diverse ‘prostaglandin-inde- three species: human, rats and bovine (Table VII). pendent’ effects which may contribute to their This effect is not due to the shared capacity of anti-inflammatory properties [16, 32]. Recently, these drugs to inhibit COX-2 dependent prosta- effects of currently available anti-inflammatory glandin production in these cells, since neither drugs on nitric oxide production have emerged as indomethacin nor glucocorticoids (at concen- an additional property of these agents. For trations sufficient to inhibit PGE2 production) example, we have reported [27] that salicylates, but inhibited NO release. This particular observation not indomethacin, inhibit NO production in is in contrast with the capacity of glucocorticoid LPS-stimulated murine macrophages. In those to inhibit iNOS expression in variety of other studies, aspirin consistently inhibited the ex- cell types (e.g., macrophages, hepatocytes, and pression of iNOS protein, whereas the effects of mesangial cells) across species [28–31]. These sodium salicylate on iNOS expression was more results suggest that the beneficial effects of variable. In addition, aspirin, but not sodium glucocorticoids seen in arthritis may not salicylate, inhibited the specific activity of iNOS in involve the modulation of NO in cartilage and cell free extracts, most probably due to the direct chondrocytes. acetylation of iNOS by aspirin [27]. These data are The current data is consistent with previous consistent with those of Aeberhard, et al. [33], and results which indicate that NSAIDS, particularly Kepka-Lenhart, et al. [34] who have reported that

Table V Effect of NSAIDS on the spontaneous production of nitric oxide by rat chondrosarcoma cells in ex vivo conditions mm nitrite

Conditions 16 h 24 h 48 h 72 h Control 11.7 2 1.3 12.9 2 1.4 18.7 2 1.6 19.4 2 1.5 + L-NMA (500 mm) 3.2 2 0.4a 3.3 2 0.5a 6.5 2 0.8a 7.1 2 0.9a + Aspirin (1 mm) 10.4 2 0.4c 11.3 2 3.6c 15.4 2 0.3a 14.8 2 0.1a + Aspirin (2 mm) 9.4 2 0.4b 9.1 2 0.6b 11.8 2 0.7a 11.7 2 0.7a + Aspirin (3 mm) 7.1 2 0.6a 6.4 2 0.6a 7.8 2 0.7a 7.9 2 0.8a + Sodium salicylate (1 mm) 9.4 2 1.4c 9.9 2 1.1c 14.0 2 1.5d 15.1 2 1.5b + Sodium salicylate (2 mm) 7.9 2 0.5a 8.3 2 0.6a 11.2 2 0.6a 11.1 2 0.6a + Sodium salicylate (3 mm) 6.0 2 0.5a 6.0 2 0.3a 8.0 2 0.4a 7.9 2 0.4a + Indomethacin (20 mm) 12.6 2 0.4e 13.8 2 0.8e 20.7 2 0.8e 21.9 2 0.7e + Hydrocortisone (10 mm) 11.9 2 0.3e 12.9 2 0.3e 19.0 2 0.4e 21.3 2 0.4e Rat chondrosarcoma cells spontaneously release NO in ex vivo conditions, were incubated with various NSAIDS and NOS modulators at different concentrations at the time of seeding the cells in the medium. Nitrite levels were estimated as described in Methods. Data represent (one of the two similar expts.) mean 2 .. N = 4. The P-values described are compared with control cells devoid of any modulators. (P-value a R 0.0001, b R 0.001, c R 0.01, d R 0.1, e R 0.5). Osteoarthritis and Cartilage Vol. 6 No. 4 275 both aspirin and sodium salicylate (20 mm) inhib- important since several studies have shown that ited NO production and expression of iNOS inhibition of excess NO production in cartilage protein in murine macrophages. As was the case in may have a ‘chondroprotective role’ [10–13]. our prior studies [27], these investigators observed However, a full appreciation of the implications of that salicylates decreased iNOS protein without these findings will only be possible when the inhibiting the amount of iNOS mRNA. The precise role of both NO and PGE2 in cartilage observation that salicylates inhibit NO production homeostasis and arthritis is clearly elucidated. In at a translational step, but not at a transcriptional animal models of arthritis, inhibitors of either step, was confirmed in hepatocytes [35]. Finally, NOS or COX-2 can independently repress joint other investigators have also demonstrated that inflammation, concomitant with the attenuation of the capacity to inhibit NOS is not a property PGE2 or NO synthesis [3, 4, 39]. In general, the common to all NSAIDS. In rat cardiac fibroblasts, effects of NO on cartilage are believed to decrease for example, aspirin and sodium salicylate, but not collagen synthesis [10]; NO has also been impli- indomethacin or acetaminophen, inhibited cy- cated in promoting chondrocyte apoptosis [13]. tokine-induced nitrite accumulation in a time and PGE2 is also believed to exert predominantly dose-dependent manner (IC50 0 1mm) [36]. Thus, injurious effects known to exacerbate joint the consistent finding in the literature is that inflammation [40], induce IL-1b expression [41] and salicylates, but typically not indomethacin, inhibit promote metalloproteinase production [42–45].

NO production in a variety of cell types, including However, PGE2 has also been reported to increase chondrocytes, macrophages, hepatocytes and fibro- proteoglycan synthesis, suggesting a possible blasts, most likely via effects on translation. Our reparative role in cartilage [46]. It should be noted observation that tenidap is also an effective that NSAIDS (unlike the NOS inhibitor: L-NMA) inhibitor of NO production, indicates that this is inhibited both NO and PGE2 production in each of not a property of salicylates alone, and may be the chondrocyte systems that we assessed. Further- shared by other classes of anti-inflammatory more, (unlike NSAIDS) L-NMA augmented PGE2 agents. Our results have shown that intra-articular production in OA-affected cartilage [22] and IL-1b produced by OA-affected chondrocytes up- bovine chondrocytes stimulated in vitro. Such regulates endogenous NO production which can be studies underscore the complexity of the regu- blocked by type I soluble IL-1bR (but not soluble lation of NOS and COX-2 in tissues, and indicate TNFaR). Spontaneous production of NO by OA that the pharmacological interference with one cartilage can also be blocked by cytokine pathway may yield unanticipated effects upon the suppressive anti-inflammatory drugs: CSAID other. Taken together, these studies do indicate which inhibit p38 kinase [37]. Our current that drugs exhibiting selectivity towards both experiments also demonstrate that Tenidap, which NOS and COX-2 may offer the potential for is known to inhibit PGE2 production [38], and developing novel anti-inflammatory therapies with synthesis of IL-1b and IL-6 [24] also inhibits the potential to prevent progressive cartilage another pleiotropic inflammatory molecule: NO. deterioration. The possibility that the inhibition of NO production by tenidap is due to the inhibition of the Acknowledgments expression of IL-1b cannot be ruled out by our experiments. We thank Ms Una Yearwood for preparation of the It should be noted that aspirin which inhibits the manuscript. We would like to thank Pfizer, Inc., U.S. Pharmaceuticals Group, New York for financially spontaneous release of NO in OA-affected carti- supporting part of this project. lage, like tenidap, also blocked the IL-1b induced NO production in OA-affected cartilage. This is not surprising because previous studies have shown that aspirin in murine macrophages block References NOS expression at the translational level and 1. Nathan C, Xie Q. Nitric oxide synthases: role, tolls, probably also directly at the enzyme level by and controls. Cell 1994;78:915–18. acetylating the enzyme to render it inactive [27]. 2. Schmidt HHHW, Walter U. NO at work. Cell What are the implications of these observations 1994;78:919–25. that salicylates and tenidap, but not indomethacin, 3. McCArtney-Francis N, Allen JB, Mizel DE, et al. inhibit NO production by chondrocytes, particu- Suppression of arthritis by an inhibitor of nitric oxide synthase. J Exp Med 1993;78:749–54. larly that produced by OA cartilage? Differences 4. Stefanovic-Racic M, Stadler J, Evans CH. Nitric among currently available anti-inflammatory Oxide and Arthritis. Arthritis Rheum agents with regard to NOS inhibition may be 1993;36:1036–44. 276 Attur et al.: Effect of NSAIDS on NO and PGE2 in chondrocytes

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