Nitric oxide enhances cyclooxygenase activity in articular cartilage

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Inflamm Res 45:254-258 (1996)

1023-3830/96/050254-05 $1.50 + 0.20/0 9 1996 Birkhfiuser Verlag, Basel

Nitric oxide enhances cyclooxygenase activity in articular cartilage L. Manfield 1'*, D. Jang, 2 and G. A. C. Murrell 2 1The Hospital for Special Surgery, Cornell University Medical College, New York, NY, USA 2Department of Orthopaedic Surgery, University of New South Wales, The St. George Hospital, Kogarah, Sydney, NSW 2217, Australia Received 28 December 1995; returned for revision 29 January 1996; accepted by W. B. van den Berg 13 February 1996

Abstract. Nitric oxide ( N O ) is a small messenger molecule synthesized by a family of enzymes, the nitric oxide synthases. Cyclooxygenases are a group of proinflammatory enzymes that release prostaglandins including prostaglandin E2 (PGE2). Both nitric oxide synthase and cyclooxygenase are involved in the inflammatory cascade of arthritis. However, the relationship between these two enzymes and their products has not been explored in articular cartilage. Here we show that in cultured bovine chondrocytes and explants of human osteoarthritic cartilage both nitric oxide synthase and cyclooxygenase activities were induced by the inflammatory mediators, lipopolysaccharide, and interleukin-1/3 or tumor necrosis factor-c~. When nitric oxide synthase activity was inhibited, PGE2, synthesis was inhibited. N O donors also induced PGE2 synthesis and N O scavengers inhibited cyclooxygenase activity. Taken together, these results support the concept that PGE2 synthesis is directly related to NO' formation and that NO' may modulate cyclooxygenase activity in articular cartilage. Key words: Nitric oxide - Cyclooxygenase - Arthritis Chondrocyte - Cartilage

Introduction Nitric oxide ( N O ) is a very small molecule synthesized from the amino acid L-arginine by a family of enzymes, the nitric oxide synthases. Its small size and its unpaired electron (denoted ), make it a highly reactive and locally diffusible free radical. Inducible forms of nitric oxide synthase are found in phagocytic cells, hepatocytes, and in cartilage [1-3]. NO" is overproduced in osteoarthritis and rheumatoid arthritis [4]. Moreover, data from our laboratory suggests that chondrocytes are the primary

* Finalist in the 1995 Westinghouse ScienceTalent Search, the 1995 Otto Burgdorf Competition, and the 1995 St. Johns New York Symposium. Correspondence to. G. A. C. Murrell

producer of N O amongst cells normally found in articular joints [1, 5]. Prostaglandins belong to a group of bioactive compounds that modulate cellular function and are derived from a twenty carbon polyunsaturated fatty acid of the n-6 and n-3 classes. The central enzyme in the prostaglandin synthetic pathway is cyclooxygenase (COX), a membrane-bound heme which has been localized to both the endoplasmic reticulum and the nuclear membrane. COX also exists in both constitutive (COX1) and inducible (COX2) forms [6]. Cyclooxygenase is inhibited by anti-inflammatory drugs, including aspirin and sulindac [7]. Prostaglandins are extremely potent and are produced in small amounts in response to stimuli, carry out their reactions at the site of synthesis, and are degraded rapidly. Some of their effects include vasodilatation, chemotaxis and mediation of pain. In arthritis, inflammatory mediators such as interleukin-1/? (IL-ll3) and tumor necrosis factor-c~ (TNF-o0 induce COX activity in synovium and cartilage and induce degeneration of the articular surfaces [8, 9]. The same inflammatory mediators induce nitric oxide synthase in articular cartilage [2]. There is evidence in macrophages that NO' regulates COX [10]. We hypothesized that NO' also regulates COX in cartilage. The aim of these experiments, therefore, was to explore the relationship between nitric oxide synthase and cyclooxygenase and their products in articular chondrocytes and cartilage.

Methods Materials

Dulbecco's modification of Eagle's medium (DMEM), Ca 2+ and Mg2+ free Dulbecco's phosphate buffered saline (PBS) and Hank's solution, antibiotic-antimycotic solution (#600-5240; 10,000U/ml penicillin G sodium; 10,000~tg/ml streptomycin sulfate; 25~tg/ml amphotericin B), HEPES solution (238.3g/1), trypsin solution (0.25% (w/v) trypsin in Hank's solution) and fetal calf serum (FCS) were purchased from Gibco Laboratories Ltd, New York, NY, USA. Tissue culture plates were from Falcon | Becton Dickinson & Co, New York, NY, USA. Picopro| scintillation vials and UltimaGold | scintillation fluid were purchased from Packard Technologies, Downers Grove, IL, USA. Sulindac

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Nitric oxide and cartilage

(5-fluoro-2-methyl-1-(4-(methylsulfinyl)phenyl)-1H-indene-3 acetic acid) was a gift of Merck Sharp & Dohme Research Laboratories, Rathway, NJ, USA. S-nitroso-N-acetyl-D,L-penicillamine(SNAP) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassfltm salt (CPTIO) were purchased from Alexis Corporation, San Diego, CA, USA. Nitroglycerin was purchased from SoloPak Laboratories Inc., Grove Village, IL, USA. S-(2-aminoethyl)is othiouronium bromide hydrobromode (AETU) [11] was purchased from Aldrich Chemical Co, Gillingham, Dorset, UK. Tumor necrosis factor-c~ (TNF-c~) was a gift from Grace Wong, Genentech Corp, South San Francisco, CA, USA. Prostaglandin E2 (PGE2) radioimmunoassay kit was purchased from DuPont de Nemours & Co., N.E.N. Research Products, Boston, MA, USA. PGE2 enzymelinked immuno-sorbant assay (EIA) was purchased from Cayman Chemical Company, Ann Arbor, MI, USA. All other chemicals and biochemicals were purchased from Sigma Chemical Co, St. Louis, MO, USA.

255 competitive binding, where a radio-labelled or an enzyme-linked antigen competed with a non-labelled antigen for a fixed number of antibody binding sites. Unlabeled antigen from standards or samples (100 gl media for the RIA assay and 50 gl for the EIA assay) were utilized and the assay performed as per manufacturers instructions. Results obtained for the standards were used to construct a standard (dose-response) curve from which the unknowns were calculated by interpolation. The sensitivity of these assays are approximately 10 pg/ml PGE2.

Statistical analysis Statistical analysis was performed using two-tailed Student's t-tests.


Sample collection and culture conditions The study was approved by the Hospital for Special Surgery Institutional Review Board. Normal human cartilage was obtained from a 7 year old girl undergoing a knee fusion for proximal femoral focal deficiency. Osteoarthritic cartilage was obtained during total knee replacement surgery in patients with osteoarthritis. Bovine cartilage was obtained from occipital articular cartilage. Each sterile tissue sample was immediately placed in cold (4~ tissue culture medium containing 90% (w/v) DMEM, 1% (v/v) antibioticantimycotic solution, 0.22% (w/v) NaHCO3 with 10% (v/v) fetal calf serum, pH 7.35. The cartilage was cleaned of non-cartilaginous tissue, washed in sterile normal saline (NS) (x6), 10% (v/v) antibiotic-antimycotic solution in Hank's (x2), and NS (x 1). For explant culture, 4 mm diameter cartilage disks were made using an 4 mm skin biopsy punch (SMS Instruments, Columbia, MD, USA) and placed with 250 gl media (DMEM with 10% FCS) into each well of a 96-well tissue culture, cultured at 37 ~ and utilized within 24 h. Chrondrocytes were obtained by collagenase digestion [12] of slices of bovine articular occipital cartilage in 0.025% (w/v) collagenase, 1% (v/v)antibiotic-antimycotic solution, 2% (v/v) HEPES solution with gentle agitation at 37~ for 16h. The cell suspension was counted, then spun at 30,000 rpm for 15 rain, the supernatant discarded, the cells washed in 10% (v/v) antibioticantimycotic solution and plated in 75 ml culture fasks at 2 x 106 cells/flask in DMEM with 10% FCS. When confluent (usually 7-10 days), the cells were trypsinized into 96 well plates at 105 cells/well for nitrite and PGE2 assays. All cultured cells were primary passage.

Nitrite release Nitrite (NO]), a stable end-product of nitric oxide, was measured in the media of cultured cells and explants utilizing the spectrophotometric method based on the Greiss reaction [13]. The absorbance was measured at 550/650nm with a 340ATTC microplate photometer (Tecan US Inc., Research Triangle Park, NC, USA). The sensitivity of this assay is approximately 1 nmol NO2-.

Cell viability I n all e x p e r i m e n t s cell viability was tested utilizing M T T . No treatment inhibited mitochondrial MTT conversion to formizan.

PGEe activity P r o s t a g l a n d i n E2 (PGE2) was m e a s u r e d in the supern a t a n t o f c u l t u r e d b o v i n e c h o n d r o c y t e s . Bovine c h o n d r o cytes in D M E M synthesized 5 - 1 0 ng PGE2/106 cells/24 h. PGE2 synthesis increased when the cells where treated with T N F - a , ILl-/3 o r E. coli polysaccharide (LPS) (Fig. 1). S u l i n d a c ( 1 0 - S M ) , a n o n steroidal a n t i - i n f l a m m a t o r y agent which blocks cyclooxygenase, i n h i b i t e d P G E 2 synthesis by c u l t u r e d c h o n d r o c y t e s s t i m u l a t e d with L P S to less t h a n 2 ng PGE2/106 cells/24 h (Fig. 1). E x p l a n t s o f o s t e o a r t h r i t i c h u m a n cartilage released 160 pg P G E 2 / 4 m m plug/24 h. W h e n s t i m u l a t e d with 30

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Cell viability Cell respiration, an indicator of cell viability, was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) to formazan [14].

Prostaglandin E 2 assays PGE 2 was assessed in the media (DMEM without FCS) of cells or explants at 24 h. The basic principle of both the radioimmunoassay (RIA) and the enzyme-linked immunosorbant assay (EIA) were



Fig. 1. Prostaglandin E2 (PGE2) release by cultured bovine articular chondrocytes was induced by 10gg/ml E. coli polysaccharide (LPS). This induction was inhibited by a 10- 5 M sulindac (Su), a cyclooxygenase inhibitor. N = 6 for each group; mean 5: SEM, *= p < 0.05 as compared with control using unpaired twoway Student's t-tests.


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Fig. 2. Induction of nitrite (NO2), the stable end-product of N O occurred in a dose-response manner when chondrocytes were exposed to LPS, TNF-a or IL-lfl. In each case, nitrite formation was completely inhibited by 10 3M aminoguanidine (*), a competitive inhibitor of nitric oxide synthase. The induction of nitric oxide synthase activity was also inhibited to 20gg/ml cycoheximide (**), an inhibitor of protein synthesis. N = 6 for each group; mean 4- SEM, *** = p < 0.001 as compared with control using un-paired two-way Student's t-tests,

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Fig. 3. Endogenous nitric oxide modulates cyclooxygenase activity (as measured by PGE2 synthesis) in cultured bovine chondrocytes. Induction of endogenous N O was achieved with 10gg/ml lipopolysaccharide (LPS), 100ng/ml tumor necrosis factor-a (TNF-a) and 100ng/ml interleukin-1/3 (ILl-/3). Nitric oxide synthase was inhibited with 5 • 10.5 M S-(2-aminoethyl)isothiouronium bromide hydrobromide (AETU). N = 6 for each group; mean 4-SEM, * = p < 0 . 0 5 , * * = p < 0 . 0 1 as compared with control using un-paired two-way Student's t-tests. 100ng/ml TNF-oz, PGE2 synthesis increased to 175pg P G E 2 / 4 m m plug/24 h (p < 0.05).

Nitric oxide synthase activity The ability for cultured bovine c h o n d r o c y t e s to release






(b) Fig. 4. Nitric oxide synthase and cyclooxygenase activity in cultured human osteoarthritic cartilage, a) Nitrite (NO2) release, b) prostaglandin E2 in media at 24 h. Note that NO~ production and cyclooxygenase activity were stimulated by TNF-c~ (100 ng/ml tumor necrosis factor-a) and this stimulation was inhibited by 10-4M of the nitric oxide synthase inhibitor, S-(2-aminoethyl) isothiouronium bromide hydrobromide (AETU). N = 14 for each group; mean4-SEM. *= p < 0.05 as compared with control * * * = p < 0.001 compared with TNF-a alone using un-paired two-way Student's t-test. nitric oxide (NO-) was d e t e r m i n e d by m e a s u r i n g the c o n c e n t r a t i o n o f nitrite ( N O 2 ) , a stable end p r o d u c t o f nitric oxide, in the s u p e r n a t a n t o f c u l t u r e d bovine c h o n d r o c y t e m o n o l a y e r s . W h e n s t i m u l a t e d with LPS, TNF-oz o r ILl-/3, c u l t u r e d bovine c h o n d r o c y t e s released

Vol. 45, 1996

Nitric oxide and cartilage



A r.


"3 0







Fig. 5. Exogenous nitric oxide modulates cyclooxygenaseactivity.

Exogenous NO was generated by 10-4M S-nitroso-N-acetyl-D,Lpenicillamine(SNAP). NO was scavengedby 10 -3 M (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxidepotassium salt (CPTIO). N = 6 for each group; mean 4- SEM, * = p < 0.05, 9* = p < 0.01 as compared with control using un-paired two-way Student's t-tests. up to 250nmol NOy/106 cells/24h. The induction of NO2 was dose-dependant (Fig. 2) and time dependant (not shown) with maximal stimulation at 24h. The induction of NO~ was inhibited by cycloheximide, indicating that de novo protein synthesis was necessary for NO2 formation. NO~ formation was inhibited by the nitric oxide synthase inhibitors: aminoguanidine, Nw-nitro-Larginine methyl ester (L-NAME) and AETU (Fig. 2).

Nitric oxide-cyclooxygenase interactions Endogenous NO~- synthesis was induced by culturing bovine chondrocytes in the presence of LPS, T N F - a and IL-1/3. This combination of inflammatory mediators also induced PGE2 synthesis (Fig. 3). However, the induction of PGE2 was completely inhibited when a nitric oxide synthase inhibitor (AETU) was added to the mixture of inflammatory mediators (Fig. 3), implying that nitric oxide synthase activity was required for PGE 2 synthesis. Endogenous NO2 synthesis was induced in explants of osteoarthritic human cartilage with TNF-~. TNF-a also induced PGE2 synthesis. However, the induction of PGE 2 and NO~ synthesis was completely inhibited with AETU (Fig. 4a and b), implying that nitric oxide synthase activity was required for PGE 2 synthesis in osteoarthritic cartilage. A direct role for N O in modulating cyclooxygenase activity was confirmed by culturing bovine chondrocytes in the presence of a N O donor (SNAP) and in the presence of a N O scavenger (CPTIO). SNAP induced PGE2 synthesis, while CPTIO inhibited PGE2 synthesis (Fig. 5). Discussion

This study is the first to show that induction of bovine

chondrocyte and human osteoarthritic cartilage cyclooxygenase activity is dependent upon and mediated via nitric oxide. Once induced, nitric oxide synthase activity correlated with increased cyclooxygenase activity in chondrocyte cultures and in conditioned media from osteoarthritic human cartilage. When nitric oxide synthase activity was inhibited, cyclooxygenase activity was also inhibited. Exogenous N O also induced cyclooxygenase activity and N O scavengers inhibited cyclooxygenase activity. The mechanism whereby N O modulated cyctooxygenase activity and any cyclooxygenase isoform specificity for this mechanism is undetermined. NO' can directly interact with other metal containing enzymes to either stimulate (i.e. soluble guanylate cyclase [t5] or inhibit (e.g. aconitase [16]) the enzymatic activity. Salvemini et al. showed in a macrophage cell line that N O directly interacted with cyclooxygenase to increase the enzyme's activity [17]). Thus it is conceivable that N O directly interacted with the metal containing active site of cycooxygenase to alter the enzyme's configuration and activate the cyclooxygenases. Aeberhard et al. [18] have recently shown that in rat macrophages nonsteroidal antiinflammatory agents inhibit N O formation by inhibiting the expression of the inducible nitric oxide synthase gene. All the forms of arthritis are characterized by increases in inflammatory mediators, such as interleuldn1/3 (IL-1/3) and tumor necrosis factor-c~ (TNF-a), increases in prostaglandin formation, increases in the enzymes that degrade cartilage (the metalloproteases) and a decrease in cartilage matrix synthesis (proteoglycans and Type II collagen). We and others have shown that bacterial cell wall products such as LPS and the inflammatory mediators, IL-1/3, TNF-c~ and interferon- 7 induce nitric oxide synthase activity in chondrocytes [1-3]. NO' released from chondrocytes mediates several of the catabolic events associated with cartilage degradation, in particular, activation of metalloprotease enzymes [19] and suppression of proteoglycan synthesis [20, 21]. The results of this study indicate that N O also regulates cyclooxygenase activity in articular cartilage. N O may play a pivotal role in the cascade of arthritis. N O , therefore, represents a key molecule in the inflammatory and degradative cascade of arthritis. The L-argininenitric oxide synthase pathway may be a novel therapeutic target for the prevention and management of arthritis.

Acknowledgements. This work was supported in part by National Institutes of Health Grant 1R29AR42729, by the Soft Tissue Research Fund and by St. George Private Hospital. We are grateful to Russell Warren, MD, Jo Hannafin, MD, PhD, and Stephen Doty, PhD of the Hospital for Special Surgery and Mr Mitchell Fox, Bronx Science High School, for advice and support.


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L. Manfield, D. Jang and G. A. C. Murrell


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