Cycloamphilectenes, a new type of potent marine diterpenes: inhibition of nitric oxide production in murine macrophages

Life Sciences 72 (2003) 2543 – 2552 www.elsevier.com/locate/lifescie

Cycloamphilectenes, a new type of potent marine diterpenes: inhibition of nitric oxide production in murine macrophages Rut Lucas a, Agostino Casapullo b, Linda Ciasullo b, Luigi Gomez-Paloma b, Miguel Paya´ a,* a

Departamento de Farmacologı´a. Universidad de Valencia. Facultad de Farmacia. Av. Vicent Andre´s Estelle´s s/n, 46100 Burjasot, Valencia, Spain b Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy Received 30 May 2002; accepted 13 December 2002

Abstract The inhibitory effect of a series of 6 cycloamphilectenes, novel marine diterpenes based on amphilectene skeletons and isolated from the Vanuatu sponge Axinella sp., on NO, PGE2 and TNFa production in murine peritoneal macrophages was studied. These compounds reduced potently nitric oxide production in a concentration-dependent manner with IC50 values in the submicromolar range (0.1 – 4.3 AM). Studies on intact cells and Western blot analysis showed that the more potent cycloamphilectenes reduced the expression of inducible nitric oxide synthase without affecting cyclo-oxygenase-2 expression. Among them cycloamphilectene 2, the unique compound bearing an exocyclic methylene group, was able to reduce NO production without affecting TNFa release. Cycloamphilectene 2, which is an inhibitor of the nuclear factor-kB pathway, exhibited topical anti-inflammatory activity. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Cycloamphilectenes; Nitric oxide; iNOS; NF-kB; Macrophages; Zymosan

Introduction Prostaglandins and nitric oxide (NO) are ubiquitous mediator systems which have numerous vascular and inflammatory effects. Many cell types have the capacity to generate them. Production of

* Corresponding author. Tel.: +34-963864946; fax: +34-963864943. E-mail address: [email protected] (M. Paya´). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00167-X

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prostaglandins or NO by the constitutive isoenzymes cyclo-oxygenase-1 or endothelial NO synthase, are implicated in the physiological regulation of vascular tone and homeostatic functions. Cyclo-oxygenase2 and inducible NO synthase are not generally expressed in resting cells, but are induced following appropriate stimulation with proinflammatory agents such as cytokine, lipopolysaccharide and zymosan in numerous cell types including macrophages [17,26]. The induction of cyclo-oxygenase-2 and NO synthase results in the increased synthesis of prostaglandins and NO, which play a key role in the pathophysiology of inflammatory conditions including arthritis [14,15,29]. The participation of NO in articular degradation has been reviewed [13]. In addition, NO can enhance the production of a variety of other inflammatory mediators, including tumour necrosis factor-a and interleukin-1h which participate in the macrophage-dependent inflammatory response [18]. Nuclear factor-nB (NF-nB) comprises a family of inducible transcription factors that serve as important regulators of the host immune and inflammatory response. NF-nB has also been implicated in the pathogenesis of chronic inflammatory diseases, such inflammatory bowel diseases and rheumatoid arthritis [28]. Activation of NF-nB increases the expression of genes encoding many proinflammatory mediators, including cytokines, receptors required for cell adhesion and migration, as well as inducible enzymes such as iNOS and COX-2 [21]. Little is known about diterpenes based on amphilectene and cycloamphilectene skeletons. This type of unusual metabolites are usually characterized by poorly functionalized tri- or tetracyclic carbon frameworks, but in most cases one or more isonitrile, isocyanide or isothiocyanide functions are present. Some of them have been reported as inhibitors of proliferation of lymphocytes T and B under various stimuli [5]. The present study was undertaken to examine the effects of a series of 6 new cycloamphilectenes (Fig. 1), on murine peritoneal macrophage and human neutrophil functions as well as on several enzymes relevant to the inflammatory process. The results of this study demonstrate the in vitro inhibitory effects on cell functions exerted by these compounds. Cycloamphilectene 2 also exhibited topical anti-inflammatory activity in vivo. This is the first report concerning the pharmacological properties of these marine structures. Some structure-activity relationships considerations have been deduced.

Fig. 1. Chemical structures of cycloamphilectenes 1 – 6.

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Materials and methods Preparation of human neutrophils Leukocytes were obtained and purified as previously was described [2]. Viability was greater than 95% by the trypan blue exclusion test. The mitochondrial dependent reduction of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan [11] was used to assess the possible cytotoxic effect of cycloamphilectenes derivatives on human neutrophils. Isolation and culture of mouse peritoneal macrophages Female Swiss mice weighing 25–30 g were used to obtain highly purified peritoneal macrophages. Cells were harvested by peritoneal lavage 4 days after i.p. injection of 1 ml of 10% thioglycolate broth. Cells were resuspended in culture medium (120 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl2  7H2O, 1.2 mM KH2PO4, 25 mM NaHCO3, 10 mM Hepes, 1 mM arginine and 10 mM glucose) supplemented with 10% foetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 Ag/ml streptomycin and incubated at 37 jC for 2 h. The adherent cells were used to perform the following experiments. Cytotoxicity was assessed by the reduction of MTT [11]. Elastase release by human neutrophils Neutrophils (2.5  106 cells/ml) were preincubated with test compound or vehicle for 5 min and then stimulated with cytochalasin B (10 AM) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 10 nM). Elastase activity was estimated in supernatants as p-nitrophenol release. Possible direct inhibitory effects on elastase activity were also assessed [9]. Nitrite, prostaglandin E2 and TNFa production in mouse peritoneal macrophages Peritoneal macrophages (4  105 cells/well) were incubated with unopsonized zymosan (0.1 mg/ ml) in 96-well culture plate at 37 jC for 18 h (induction phase) in the presence of test compounds or vehicle. Nitrite, prostaglandin E2 and TNFa levels were determined in culture supernatants by a fluorimetric method [19], by radioimmunoassay [20] and by time-resolved fluoroimmunoassay [23], respectively. In another set of experiments zymosan-stimulated cells were collected to determine inducible NO synthase and cyclo-oxygenase-2 expression by Western blot analysis as described below. Inducible NO synthase and cyclo-oxygenase-2 activity in intact cell 18 h zymosan-stimulated macrophages (4  105 cells/well) were washed and fresh medium supplemented with L-arginine (0.5 mM) and arachidonic acid (10 AM) was added for a further 2 h (post-induction phase) incubation with test compounds. Supernatants were collected for the measurement of nitrite and prostaglandin E2 accumulation for the last 2 h. Nitrite concentration as reflection of NO released was assayed fluorometrically and prostaglandin E2 levels were assayed by radioimmunoassay.

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Western blot analysis To induce iNOS and COX-2 expression, murine peritoneal macrophages were incubated with zymosan (0.1 mg/ml) at a concentration of 2  106 cells/ml in 24-well culture plate in the presence of the test compound. After 18 h stimulation, medium was removed and cells were washed twice with PBS, then cells were lysed with lysis buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris pH 7.4). Lysates were centrifuged at 10,000 g for 10 min. and supernatants were used for western blot assay, 25 Ag of protein were loaded on 12% PAGE-SDS and transferred onto polyvinylidene difluoride membranes for 90 min at 125 mA. Membranes were blocked in PBS-Tween 20 containing 3% w/v BSA and incubated with anti-iNOS polyclonal antibody (1/10,000 dilution, Cayman Chemical, USA) or COX-2 polyclonal antiserum (1/1,000 dilution, Cayman Chemical, USA), respectively, followed by incubation with the respective secondary antibodies: peroxidase-conjugated goat anti-rabbit IgG (1/20,000 dilution, DAKO, Denmark) and anti-goat/sheep peroxidase-conjugated (1/ 20,000 dilution, Sigma, USA). The immunoreactive bands were visualized using an enhanced chemilumiscence system (ECL, Amersham Iberica, Madrid, Spain). Electrophoretic mobility shift assay (EMSA) Nuclear and cytosolic extracts from mouse peritoneal macrophages were prepared as described [16]. Protein was determined by the DC Bio-Rad protein reagent (Bio-Rad, CA). The double-stranded oligonucleotide containing the consensus NF-jB sequence (Promega Corp., WI, USA) was end-labelled using T4 polynucleotide kinase (Amersham Pharmacia Biotech Europe GmbH, Spain) and [g-32P]ATP, followed by purification using G-25 microcolumns (Amersham Pharmacia Biotech Europe GmbH, Spain). Incubations were performed on ice with 6 Ag of nuclear extract, 100,000 c.p.m. of labeled probe, 2 Ag poly(dI-dC), 5% v/v glycerol, 1 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, 100 mM NaCl and 10 mM Tris-HCl buffer (pH 8.0) for 15 min. Complexes were analyzed by nondenaturating 6% polyacrylamide gel electrophoresis in 0.5  Tris-borate buffer followed by autoradiography of the dried gel. Mouse ear edema All studies were performed in accordance with the Declaration of Helsinki and the European Community guidelines for the handling and use of laboratory animals. The protocols were approved by the institutional Animal Care and Use Committee. 12-O-Tetradecanoylphorbol acetate (2.5 Ag, TPA) dissolved in 20 Al acetone was applied in 10 Al volumes to both inner and outer surfaces of the right ear of Swiss mice (20–25 g). Cycloamphilectene 2 and indomethacin were applied topically in acetone 30 min before TPA administration. The animals were killed by cervical dislocation after 4 h, and equal sections of both ears were punched out and weighed. The increase in the weight of the right ear punch over the left indicated the edema [3]. Materials Cycloamphilectenes (1–6) were isolated from the apolar extract of the marine sponge Axinella sp. according to modified literature procedures [4]. The biological material was collected from the Vanuatu

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Islands (depth-14 m) and taxonomically identified by Dr. John Hooper (voucher specimen located at Museum of Queensland, Brisbane, Australia; accession number G306878). [5,6,8,11,12,14,15(n)-3H] prostaglandin E2 and [g-32P]ATP were from Amersham Iberica (Madrid, Spain). Inducible NO synthase and cyclo-oxygenase-2 specific polyclonal antisera were purchased from Cayman Chem. (MI, U.S.A). The rest of reagents were from Sigma Chem. (MO, U.S.A.). Statistical analysis The results are presented as mean F S.E.M; n represents the number of experiments. Inhibitory concentration 50% (IC50) values were calculated from at least 4 significant concentrations (n = 6). The level of statistical significance was determined by analysis of variance (ANOVA) followed by Dunnett’s t-test for multiple comparisons.

Results Production of nitrite and prostaglandin E2 in stimulated mouse peritoneal macrophages Mouse peritoneal macrophages incubated for 18 h (induction phase) with zymosan and with the cycloamphilectenes 1–6 demonstrated a greater inhibition on nitrite (as index of NO generation) production than on prostaglandin E2 production. Table 1 also shows the IC50 values of tested compounds for nitrite. As expected, dexamethasone (a potent inhibitor of inducible NO synthase and

Table 1 Inhibitory activity of cycloamphilectenes 1 – 6 on the production of nitrite and PGE2 in mouse peritoneal macrophages Non-stimulated cells

18 h treatment (induction phase) Nitrite (ng/ml)

Zymosan-stimulated cells 1 2 3 4 5 6 Dx NS-398

2 h treatment (post-induction phase) PGE2 (ng/ml)

Nitrite (ng/ml)

PGE2 (ng/ml)

65.5 F 6.8**

1.2 F 0.2**

22.3 F 3.8**

1.5 F 0.2**

416.8 F 35.2

7.1 F 0.6

125.8 F 14.1

8.3 F 1.0

% I (10 AM)

IC50 (AM)

% I (10 AM)

% I (10 AM)

% I (10 AM)

90.2 F 91.8 F 71.6 F 83.4 F 91.4 F 84.1 F 100.0 F N.D.

1.1 (0.3 – 3.9) 0.1 (0.1 – 0.3) 4.3 (2.5 – 7.0) 0.5 (0.3 – 0.6) 0.6 (0.3 – 1.3) 0.2 (0.1 – 0.4) 0.02 (0.01 – 0.03) N.D.

49.2 56.0 37.8 69.8 48.8 50.3 97.4 97.7

12.8 14.2 0.5 30.3 10.1 27.4 0.5 0.0

36.3 30.1 30.3 51.8 31.3 29.2 0.7 81.5

5.8** 4.1** 0.2** 5.0** 1.3** 3.1** 0.0**

F F F F F F F F

6.2** 5.1** 3.0** 3.6** 7.9** 4.8** 4.5** 1.0**

F F F F F F F F

5.3 5.1 0.5 4.0* 3.6 2.3* 0.5 0.0

F F F F F F F F

1.7** 2.8* 2.8* 2.8** 3.5* 6.1* 0.4 4.9**

Results show mean F S.E.M of percentages of inhibition (% I) at the concentration of 10 AM (n = 6), and IC50 (AM, with the 95% confidence limits) values were determined from at least 4 different and significant concentrations (n = 6). N.D. = Not determined. * P < 0.05 compared with zymosan-stimulated cells. ** P < 0.01.

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cyclo-oxygenase-2 gene expression) reduced nitrite and prostaglandin E2 levels. None of these compounds affected cellular viability, as assessed by mitochondrial reduction of MTT after 24 h (data not shown), indicating that they were not cytotoxic. To establish whether the inhibition of nitrite and prostaglandin E2 production was due to an interaction with the enzyme induction by zymosan or to a direct action of cycloamphilectenes 1–6 on inducible NO synthase and cyclo-oxygenase-2 activities, these compounds as well as dexamethasone were added to cells which previously had expressed inducible NO synthase and cyclo-oxygenase-2 and were then incubated for 2 h (postinduction phase) in fresh culture medium supplemented with L-arginine and arachidonic acid. Cycloamphilectenes, which produced a significant reduction in nitrite production in the induction phase (18 h treatment), did not affect to the same extent nitrite content in the post-induction phase (2 h treatment). In contrast, cycloamphilectenes produced a significant inhibition of prostaglandin E2 production in both experimental conditions. As expected, dexamethasone significantly modified the levels of both metabolites in the induction phase (18 h treatment). The selective cyclo-oxygenase-2 inhibitor NS-398 caused a very significant reduction of prostaglandin E2 production. This suggests that cycloamphilectenes could display a dual inhibitor profile, acting as inducible NO synthase expression inhibitors in addition to directly inhibiting prostaglandin E2 production at the level of cyclo-oxygenase-2. Production of TNFa in stimulated mouse peritoneal macrophages Mouse peritoneal macrophages incubated for 18 h zymosan and cycloamphilectenes 1–6 at 1 and 10 AM caused concentration-dependent inhibition on TNFa production for all the compounds studied except for cycloamphilectene 2 which was inactive (Fig. 2). Inducible NO synthase and cyclo-oxygenase-2 protein expression in mouse peritoneal macrophages Western blot assays of 18 h zymosan-stimulated cells were performed with those cycloamphilectenes (2, 4 and 6) which showed a higher potency (IC50 values of 0.1, 0.5 and 0.2 respectively) reducing nitrite

Fig. 2. Inhibitory activity of cycloamphilectenes 1 – 6 on the production of TNFa in murine peritoneal macrophages. B: nonstimulated cells. C: zymosan-stimulated cells. Data represent mean F S.E.M., n = 6 – 10. *P < 0.05, **P < 0.01.

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Fig. 3. Effect of cycloamphilectenes 2, 4 and 6 on inducible NO synthase and cyclo-oxygenase-2 expression in murine peritoneal macrophages. The figure is representative of two similar experiments. B: non-stimulated cells. C: zymosanstimulated cells. 2: zymosan-stimulated cells treated with cycloamphilectene 2 at 1 AM. 4: zymosan-stimulated cells treated with cycloamphilectene 4 at 1 AM. 6: zymosan-stimulated cells treated with cycloamphilectene 6 at 1 AM. Dx: zymosan-stimulated cells treated with dexamethasone at 1 AM.

production, to assess possible effects on inducible NO synthase or cyclo-oxygenase-2 protein expression. Fig. 3 shows a representative experiment where co-incubation of cycloamphilectenes at 1 AM with zymosan (0.1 mg/ml) for 18 h, caused a partial reduction of inducible NO synthase expression for these cycloamphilectenes without any effect on cyclo-oxygenase-2 expression. As expected, dexamethasone potently inhibited the expression of both enzymes. Effect on zymosan-induced NF-jB-DNA binding activity To evaluate if cycloamphilectene 2 may act on the NF-nB pathway, we analyzed nuclear protein extracts from mouse peritoneal macrophages stimulated with zymosan either in the presence or absence of this compound, for NF-nB-DNA binding activity using a radiolabeled NF-nB-specific oligonucleotide. Strong radioactive DNA binding to nuclear proteins was observed after 2 h when cells were treated with zymosan. Nuclear extracts of cells incubated with cycloamphilectene 2 and zymosan showed a protein-DNA complex migrating at the same mobility but the DNA-binding activity was reduced as compared to the zymosan control at concentration of 10 AM (Fig. 4). The proteasome inhibitor MG132, used as reference compound, weakly inhibited NF-nB-DNA binding activity.

Fig. 4. Effect of cycloamphilectene 2 on NF-nB-DNA binding in nuclear extracts of mouse peritoneal macrophages. Cells were preincubated with cycloamphilectene 2 (1 and 10 AM) or MG132 (1 AM) for 30 min before zymosan stimulation for 2 h and then nuclear extracts were obtained as described under Material and methods. B: basal (non-stimulated cells): Z: zymosan. Results are representative of two independent experiments.

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Fig. 5. Topical effect of cycloamphilectene 2 and indomethacin on the mouse ear edema model induced by TPA. Data represent mean F S.E.M., n = 6. **P < 0.01 with respect to TPA control group. Test compounds (Ag/ear) were applied topically in acetone 30 min before TPA administration.

Effect on mouse ear edema Cycloamphilectene 2 inhibited potently mouse ear edema induced by TPA, after topical administration at concentrations of 100 and 200 Ag/ear (Fig. 5). The effect was dose-related with a similar potency than that of indomethacin.

Discussion The induction of NO synthase and cyclo-oxygenase-2 greatly increases the synthesis of NO and prostaglandins, which contribute to the pathophysiology of different inflammatory processes. In addition, NO has been implicated in septic shock [6]. Inducible NO synthase inhibition attenuates inflammatory response in different experimental models such as granuloma formation by subcutaneous implantation in rats of carrageenin-soaked sponges [12] and the zymosan air pouch model in mice [24]. In humans, the production of NO by activated macrophages or neutrophils can be an index of bronquial inflammation and a mechanism for amplifying asthmatic inflammation [1]. Furthermore, NO has been shown to increase the production of pro-inflammatory prostaglandins in in vitro and in vivo studies [25,26]. On the other hand, overproduction of prostaglandins by cyclo-oxygenase-2 expression in vivo has been reported in chronic inflammatory conditions such as rheumatoid arthritis [14] and experimental models of inflammation [27,29]. In this study we have shown that cycloamphilectenes inhibited the production of NO and prostaglandin E2 in murine peritoneal macrophages stimulated by zymosan without any evidence of a cytotoxic effect. Nevertheless, these compounds were practically ineffective when inducible NO synthase was already expressed. Western blot analysis of lysated mouse peritoneal macrophages showed that inducible NO synthase protein expression was reduced by the presence of these cycloamphilectenes during zymosan treatment, indicating that these compounds inhibit the induction rather than the activity of that enzyme. On the contrary, the reduction in PGE2 content, mediated by cycloamphilectenes was a consequence of the enzymatic inhibition of cyclo-oxygenase-2. It is noteworthy that among the marine diterpenes studied cycloamphilectene 2, showed a characteristic

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pharmacological profile. In this regard, this compound, which is the only one that demonstrated a great potency in inhibiting NO production, as well as the degranulation process (measured as elastase release in human neutrophils. IC50 = 3.2 AM), did not affect TNFa production. From a chemical point of view, only cycloamphilectene 2 bears an exocyclic methylene group in its structure. The potential effect on TNFa production of the rest of cycloamphilectenes could be a consequence of their action at the level of release of TNFa from the cell surface by TNFa converting enzyme (TACE), but this aspect remains to be determined. Transcription factors belonging to the NF-nB family are implicated in the expression of many inflammatory genes and inhibition of this transcription factor is associated with anti-inflammatory effects in animals models of arthritis [7,22]. Macrophages are the major cytokine producers in chronic inflammatory conditions. We have shown in murine macrophages that cycloamphilectene 2, which exerted potent topical anti-inflammatory activity, inhibits NF-nB DNA-protein binding, offering a possible mechanism for the inhibition on inducible NO synthase expression. Drugs such as glucocorticoids, able to inhibit inducible NO synthase and cyclo-oxygenase-2 expression are potent anti-inflammatory agents [8,10]. Our results indicate that cycloamphilectenes can control NO and prostaglandin E2 overproduction by dual inhibition of the enhanced inducible NO synthase expression and the cyclo-oxygenase-2 activity, in addition with their effect on TNFa production. In this respect, the use of drugs possessing distinct mechanisms of actions may have relevance to inhibition of the inflammatory response, thus representing a new approach for the modulation of different inflammatory pathologies and could be an important strategy to obtain promising anti-inflammatory agents. Acknowledgements This work was supported by grant from CICYT (SAF2001-2639), Spanish Ministerio de Ciencia y Tecnologı´a. R. Lucas was the recipient of a scholarship from FPI program.

References [1] Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. European Respiratory Journal 1993;6(9):1368 – 70. [2] Bustos G, Ferra´ndiz ML, Sanz MJ, Paya´ M, Alcaraz MJ. A study of the novel anti-inflammatory agent florifenine. Topical anti-inflammatory activity and influence on arachidonic acid metabolism and neutrophil functions. Naunyn-Schmiedeberg’s Archives of Pharmacology 1995;351(3):298 – 304. [3] Carlson RP, O’Neill-Davis L, Chang J, Lewis AJ. Modulation of mouse ear edema by cyclooxygenase and lipoxygenase inhibitors and other pharmacologic agents. Agents and Actions 1985;17(2):197 – 204. [4] Ciasullo L, Cutignano A, Casapullo A, Puliti R, Mattia CA, Debitus C, Riccio R, Gomez-Paloma L. A new cycloamphilectene metabolite from theVanuatu sponge Axinella sp. Journal of Natural Products 2002;65(8):1210 – 2. [5] Ciavatta ML, Fontana A, Puliti R, Scognamiglio G, Cimino G. Structures and absolute stereochemistry of isocyanide and isothiocyanate amphilectenes from the Caribbean sponge Cribochalina sp. Tetrahedron 1999;55(43):12629 – 36. [6] Cobb JP, Danner RL. Nitric oxide and septic shock. The Journal of the American Medical Association 1996;275(15): 1192 – 6. [7] Deng GM, Verdrengh M, Liu ZQ, Tarkowski A. The major role of macrophages and their product tumor necrosis factor alpha in the induction of arthritis triggered by bacterial DNA containing CpG motifs. Arthritis and Rheumatism 2000;43(10):2283 – 9.

2552

R. Lucas et al. / Life Sciences 72 (2003) 2543–2552

[8] Di Rosa M, Radomski M, Carnuccio R, Moncada S. Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages. Biochemical and Biophysical Research Communications 1990;172(3):1246 – 52. [9] Escrig V, Ubeda A, Ferra´ndiz ML, Darias J, Sa´nchez JM, Alcaraz MJ, Paya´ M. Variabilin: a dual inhibitor of human secretory and cytosolic phospholipase A2 with anti-inflammatory activity. Journal of Pharmacology and Experimental Therapeutics 1997;282(1):123 – 31. [10] Goppelt-Struebe M. Molecular mechanisms involved in the regulation of prostaglandin biosynthesis by glucocorticoids. Biochemical Pharmacology 1997;53(10):1389 – 95. [11] Gross SS, Levi R. Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. Journal of Biological Chemistry 1992;267(36):25722 – 9. [12] Iuvone T, Carnuccio R, Di Rosa M. Modulation of granuloma formation by endogenous nitric oxide. European Jounal of Pharmacology 1994;265(1 – 2):89 – 92. [13] Jang D, Murrell GA. Nitric oxide in arthritis. Free Radical Biology and Medicine 1998;24(9):1511 – 9. [14] Kang RY, Freire Moar J, Sigal E, Chu CQ. Expression of cyclooxygenase-2 in human and an animal model of rheumatoid arthritis. British Journal of Rheumatology 1996;35(8):711 – 8. [15] Kaur H, Halliwell B. Evidence for nitric oxide-mediated oxidative damage in chronic inflammation. Nitrotyrosine in serum and synovial fluid from rheumatoid patients. FEBS Letters 1994;350(1):9 – 12. [16] Lopez-Collazo E, Hortelano S, Rojas A, Bosca L. Triggering of peritoneal macrophages with IFN-alpha/beta attenuates the expression of inducible nitric oxide synthase through a decrease in NF-kappaB activation. Journal of Immunology 1998;160(6):2889 – 95. [17] MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annual Review of Immunology 1997;15: 323 – 50. [18] Marcinkiewicz J, Grabowska A, Chain B. Nitric oxide up-regulates the release of inflammatory mediators by mouse macrophages. European Journal of Immunology 1995;25(4):947 – 51. [19] Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG. A fluorometric assay for the measurement of nitrite in biological samples. Analytical Biochemistry 1993;214(1):11 – 6. [20] Moroney MA, Alcaraz MJ, Forder RA, Carey F, Hoult JRS. Selectivity of neutrophil 5-lipoxygenase and cyclo-oxygenase inhibition by an anti-inflammatory flavonoid glycoside and related aglycone flavonoids. Journal of Pharmacy and Pharmacology 1988;40(11):787 – 92. [21] Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 1999;18(49):6853 – 66. [22] Palombella VJ, Conner EM, Fuseler JW, Destree A, Davis JM, Laroux FS, Wolf RE, Huang J, Brand S, Elliott PJ, Lazarus D, McCormack T, Parent L, Stein R, Adams J, Grisham MB. Role of the proteasome and NF-kappaB in streptococcal cell wall-induced polyarthritis. Proceedings of the National Academy of Sciences of the United States of America 1998; 95(26):15671 – 6. [23] Pennanen N, Lapinjoki S, Palander A, Urtti A, Monkkonen J. Macrophage-like RAW 264 cell line and time-resolved fluoroimmunoassay (TRFIA) as tools in screening drug effects on cytokine secretion. International Journal of Immunopharmacology 1995;17(6):475 – 80. [24] Posadas I, Terencio MC, Guille´n I, Ferra´ndiz ML, Coloma J, Paya´ M, Alcaraz MJ. Co-regulation between cyclo-oxygenase-2 and inducible nitric oxide synthase expression in the time-course of murine inflammation. Naunyn-Schmiedeberg’s Archives of Pharmacology 2000;361(1):98 – 106. [25] Salvemini D, Manning PT, Zweifel BS, Seibert K, Connor J, Currie MG, Needleman P, Masferrer JL. Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors. Journal of Clinical Investigation 1995;96(1):301 – 8. [26] Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie MG, Needleman P. Nitric oxide activates cyclooxygenase enzymes. Proceedings of the National Academy of Sciences of the United States of America 1993;90(15):7240 – 4. [27] Seibert K, Zhang Y, Leahy K, Hauser S, Masferrer J, Perkins W, Lee L, Isakson P. Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proceedings of the National Academy of Sciences of the United States of America 1994;91(25):12013 – 7. [28] Tak PP, Firestein GS. NF-kappaB: a key role in inflammatory diseases. Journal of Clinical Investigation 2001;107(1): 7 – 11. [29] Vane JR, Mitchell JA, Appleton I, Tomlison A, Bishop-Bailey D, Croxtall J, Willoughby DA. Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation. Proceedings of the National Academy of Sciences of the United States of America 1994;91(6):2046 – 50.