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MODULATION OF PGE2 AND TNFα BY NITRIC OXIDE IN RESTING AND LPS-ACTIVATED RAW 264.7 CELLS
doi:10.1006/cyto.2002.1955, available online at http://www.idealibrary.com on
MODULATION OF PGE2 AND TNF BY NITRIC OXIDE IN RESTING AND LPS-ACTIVATED RAW 264.7 CELLS Cecilia Guastadisegni,1 Alessia Nicolini,2 Maria Balduzzi,3 Maria Antonietta Ajmone-Cat,2 Luisa Minghetti2 Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNF play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNF synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNF release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE2 production, while the LPS-stimulated production was slightly increased only by PAPA/ NO. The modulation of PGE2 synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules. 2002 Elsevier Science Ltd. All rights reserved.
Macrophages play a major role in host defence against infection and cancer. Activation of macrophages by lipopolysaccharide (LPS) enhances the production and release of inflammatory mediators, including cytokines, arachidonic acid metabolites and From the 1Laboratory of Environmental Hygiene, 2Neurobiology Section, Laboratory of Pathophysiology, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome and 3Toxicology Biochemical Science Section, ENEA, Via Anguillarese 301, Rome, Italy Correspondence to: Cecilia Guastadisegni, Laboratorio di Igiene Ambientale, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Roma, Italy. Fax: + +39-6-49387083; Tel: + +39-649902885; E-mail: [email protected] Received 6 October 2001; received in revised form 8 June 2002; accepted for publication 19 June 2002 1043–4666/02/$-see front matter 2002 Elsevier Science Ltd. All rights reserved. KEY WORDS: nitric oxide/nitric oxide-releasing compounds/ prostaglandins/Tumor Necrosis Factor/cyclooxygenase/arachidonic acid CYTOKINE, Vol. 19, No. 4 (21 August), 2002: pp 175–180
nitric oxide (NO). Nitric oxide has been shown to be involved in numerous biological effects, both physiological and pathological, and to exert several regulatory functions. The effects exerted by NO may be either direct or mediated by the reactive nitrogen oxide species (RNOS) formed in an aerobic biological environment. Indeed, NO react with oxygen, superoxide and transition metals, generating NOx, peroxynitrite and heme complexes, respectively.1 The products of these reactions can in turn support additional nitrosative reactions with nucleophilic cellular molecules.2 Among the several macrophage functions that might be regulated by NO are the syntheses of Tumor Necrosis Factor (TNF) and prostaglandins (PGs), which are important mediators of inflammation and cellular immune response. TNF, originally defined by its antitumoral activity, is now recognized as a potent mediator of 175
176 / Guastadisegni et al.
inflammatory responses possessing antibacterial and antiviral properties.3 Positive and negative regulation of TNF synthesis by NO has been reported, depending on the NO donor used.4–6 Prostaglandins, and in particular PGE2, is regarded as a potent pro-inflammatory molecule, however, increasing evidence indicated that they can also exert anti-inflammatory functions which are important for the resolution of the inflammatory response.7 They are metabolites of arachidonic acid (AA) through the cyclooxygenase (COX) metabolic pathway. As for NOS, COX exists in a constitutive isoform, which is expressed in most tissue and is involved at the maintenance of several physiological functions, and in an inducible isoform (COX-2), which is the major isoform expressed in activated inflammatory cells. Since COX is a heme protein, it has been proposed that NO enhances COX activity by interacting with the heme group, as described for the enzyme guanylate cyclase, one of the major target of NO.8 However, both positive and negative regulation of PG synthesis by NO have been reported.8–11 As suggested by Wink and Mitchell,1 one possible explanation of these conflicting results is that NO might affect COX activity by other mechanisms, in alternative to the interaction with the metal centers of the enzyme. In line with this view, we have previously demonstrated that NO modulates prostanoid synthesis by acting at different steps in the PGs synthetic pathway, including AA release and expression of COX-2.9,12 The apparent conflicting results reported in the literature on NO regulatory activities may also be related to the different experimental conditions and in particular to the specific source of NO used. Recently a group of compounds, NONOates has been introduced, which do not require enzymatic metabolism to release NO and release predictable amounts of NO at a chemically defined rate, allowing well-controlled biological studies.13,14 In the present study, we have used PAPA/NO, with a half-life of 15 min, and DETA/NO, characterised by a half-life of 20 h, to expose the cell cultures either to high levels of NO for brief time (PAPA/NO) or to low levels of NO for long time (DETA/NO). By this experimental approach we have found that level and time of exposure to NO are both crucial in determining the molecular target as well as the final result of the interactions between NO and inflammatory molecules.
RESULTS Effects of NONOates on TNF synthesis NONOates are nucleophilic complexes of NO, spontaneously releasing approximately 2 moles of NO per molecule of starting material after addition to
CYTOKINE, Vol. 19, No. 4 (21 August, 2002: 175–180)
Figure 1. Effect of PAPA/NO and DETA/NO on basal TNF production in RAW 264.7 cells. The cells were incubated in the presence of the NOS inhibitor NMMA (400 M) with or without DETA/NO (deta) and PAPA/NO (papa) at two different concentrations 0.5 and 1 mM for 5 h, 16 h and 24 h. Values are meansS.E.M. of three independent experiments assayed in duplicate. *P<0.025, **P<0.005, ***P<0.001, ****P<0.0005 compared with the corresponding NMMA treated cells.
aqueous solutions at neutral pH. Given the half-lives of PAPA/NO (t Y 15 min) and DETA/NO (t Y 20 h), the addition to the culture media of the same amount of these two NONOates results in exposure of cells to different levels of NO. Indeed, 1 mM PAPA/NO generates an immediate peak of 22 M NO, that after 1 h declines to around 1–2 M.15 On the other hand, DETA/NO, with the predicted half life of 20 h, releases a much lower but long lasting NO output.16 Cell cultures were incubated for 24 h with individual NONOates without or with LPS. In order to avoid interference with the endogenously produced NO, the effects of NONOates were studied in the presence of the NO-synthase inhibitor NG-monomethyl-L-arginine (NMMA) (400 M), able to inhibit by over 70% NO production in RAW cells.9 At the end of the incubation time, supernatants were collected and analysed for TNF contents. RAW 264.7 cells spontaneously released detectable levels of TNF after 5 h of incubation (8416 pg/ ml n=3), which further increased at 16 h of incubation (23454 pg/ml n=3) and remained stable at 24 h (21856 pg/ml n=3). The presence of 1 g/ml of LPS remarkably increased the release of TNF, which reached maximal levels between 5 h and 16 h of incubation (179324 pg/ml and 2037118 pg/ml, after 5 h and 16 h respectively, n=3). In basal conditions, PAPA/NO and DETA/NO, at two different concentrations (0.5 and 1 mM) had opposite effects on TNF release. Indeed, we found that DETA/NO decreased the basal TNF release at 16 h and 24 h, whereas PAPA/NO caused an increase in the synthesis of TNF, already detectable after 5 h of incubation with the higher concentration of the
Effect of nitric oxide on PGE2 and TNF- in RAW 264.7 cell / 177
Figure 2. Effect of PAPA/NO and DETA/NO on basal and LPS induced PGE2 production in RAW 264.7 cells. Cells were incubated with or without 1 g/ml LPS in the presence of the NOS inhibitor NMMA (400 M) with or without DETA/NO (deta) and PAPA/NO (papa) at two different concentrations 0.5 and 1 mM for 24 h. One experiment assayed in triplicate representative of four is shown.
NONOate (Fig. 1). Conversely, PAPA/NO did not affect LPS-induced TNF release at any concentration and at any experimental time point, while DETA/NO slightly reduced the stimulation of TNF at 16 h (data not shown).
Effects of NONOates on PGE2 synthesis The effect of the two NO-donors on PG synthesis was evaluated in the same experimental conditions described above. In Figure 2, one representative experiment of four is shown. Both NONOates dosedependently increased basal PGE2 production. DETA/NO increased PGE2 by 19460% and 23464% (n=4) of control levels at 0.5 mM and 1 mM respectively. Similarly, PGE2 production in the presence of PAPA/NO was 17417% and 23868% (n=4) at 0.5 mM and 1 mM, respectively. The presence of 1 g/ml of LPS induced a significant increase in PGE2 accumulation (287 pg/ml and 572380 pg/ml, in basal and LPS-stimulated RAW 264.7, respectively, n=4), consistently with previous studies.9 LPS-stimulated PGE2 production was increased in the presence of PAPA/NO, while DETA/NO had no effect on this production. The presence of PAPA/NO consistently increased the LPSinduced PGE2 synthesis of 13413% (P<0.05, n=4) at 0.5 mM and 15311% (P<0.005, n=4) at 1 mM concentration.
Effects of NONOates on AA release and COX-2 expression To investigate the possible effect of the two compounds on the release of PGs precursor AA, cells were prelabelled with [3H]AA and then incubated for 2 h
and 5 h with PAPA/NO and DETA/NO, without or with 1 g/ml of LPS. As previously described the two NONOates affected in a different way the [3H]AA release in RAW 264.7 cells (Table 1). PAPA/NO (t Y 15 min) increased both basal and LPS-induced [3H]AA release after 5 h of incubation, whereas after 2 h it was ineffective on unstimulated cells, and decreased the AA-release from LPS-stimulated cells. At variance, DETA/NO (t Y 20 h) showed only a transient increase after 2 h in the presence of LPS (Table 1). We then analysed the expression of COX-2 in unstimulated and LPS-stimulated RAW 264.7 cells, in the presence of the two NONOates (Fig.3). As previously showed,9 COX-2 expression was clearly detectable in unstimulated RAW 264.7 cells, which was further enhanced by the exposure to LPS (1 g/ml). When RAW 264.7 cells were incubated for 24 h with PAPA/NO or DETA/NO, COX-2 expression was moderately increased. On the other hand, DETA/NO showed a much stronger ability to increase COX-2 expression in LPS-stimulated cells than PAPA/NO (Fig. 3).
Cell viability Incubation of RAW264.7 cells with PAPA/NO either with or without LPS had little or no effect on cell viability as measured by neutral red uptake at 24 h. However incubation with DETA/NO at 0.5 mM and 1 mM significantly reduced neutral red uptake and cellular viability or activation at 24 h (6610%, n=3 and 5411%, n=3 P<0.05; respectively). Concurrent incubation of cells with DETA/NO and LPS did not result in any greater decrease in neutral red uptake as compared to DETA/NO only (not shown).
DISCUSSION Cytotoxic and cytoprotective actions of NO have been reported. This paradox lies on NO reactivity and on its ability to react directly or indirectly with several active groups.17 With the use of selected NO donors, characterised by different half lives and kinetics of NO release, we showed that level and time of exposure are crucial in determining the effect of NO on PGE2 and TNF macrophage synthesis. Our results indicate that TNF is downregulated in RAW 264.7 cells exposed to low level of NO for long time, while is augmented when cells are exposed to high level of NO for short time. Previous studies on RAW 264.7 and J774 macrophages exposed to SIN-1, a donor of the two radicals O2and NO, which in turn combine to form peroxynitrite,2 showed a decrease in the synthesis of TNF compared to control cultures.4,5 As formation of peroxynitrite within the cells is likely to occur in the presence of low concentration
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CYTOKINE, Vol. 19, No. 4 (21 August, 2002: 175–180)
TABLE 1. Effect of PAPA/NO and DETA/NO on basal and LPS-induced [3H]AA release in RAW 264.7 cells. % [3H]AA release 2h
Control PAPA/NO 0.5 mM PAPA/NO 1 mM DETA/NO 0.5 mM DETA/NO 1 mM
5h
Control
LPS 1 g/ml
Control
LPS 1 g/ml
100 1043.2 1014.1 1045.9 11610
1307.2 813.1 813.0 14015.4 15510.6*
100 1204.8* 1346.7* 1033.8 998.1
1265.8 1476.7* 1729.5* 945.6 985.5
Cells were prelabelled with [3H]AA and then incubated for 2 h and 5 h with PAPA/NO and DETA/NO (0.5 and 1 mM) with or without 1 g/ml of LPS, in the presence of NMMA. The radioactivity released by untreated cells was taken as 100%. *P<0.05 compared with the corresponding control.
of NO,1 the effect of DETA/NO, responsible for low and prolonged NO release may be mediated by peroxynitrite. On the other hand, other studies on J774 cells showed an increased TNF synthesis upon exposure to NO, using S-nitroso-N-acetylpenicillamine (SNAP) as NO donor,18 consistently with our data in the presence of PAPA/NO. The mechanism by which NO results in these multiple effects is not known. NO can bind superoxide anion with extremely high affinity and submicromolar amounts of NO, like in the presence of DETA/NO, are likely to bind superoxide.1,19 NO has been shown to activate the TNF promoter trough the binding in Sp1 site.20 This region is therefore NO responsive and it may not respond to peroxynitrite, this may explain the difference between the two NONOates. Indeed, we
Figure 3. Effect of PAPA/NO and DETA/NO on basal and LPSstimulated COX-2 expression in RAW 264.7 cells. Cells were incubated for 24 h in complete medium, without (panel A) or with (panel B) 1 g/ml LPS in the presence of NMMA and with DETA/NO (deta) and PAPA/NO (papa) at two different concentrations 0.5 and 1 mM. Cell lysates were prepared and equal amounts of protein (25 g) were analysed by western blot using anti COX-2 specific antibodies (1:500). Protein bands were visualised using horseradish peroxidase-conjugated secondary antibodies and ECL. One experiment representative of three is shown.
showed that the two NONOates have similar effects on PGE2 synthesis, which was increased in unstimulated cultures and unaffected in LPS-stimulated cultures. However, the activities of the two NONOates were the results of distinct mechanisms. The enhancement of basal PGE2 production observed in RAW 264.7 cells after exposure to PAPA/NO seemed related to both an increased availability of the PGE2 precursor AA, and to a slight increase in COX-2 expression. In the LPSstimulated cultures the effect on AA was not sufficient to enhance PGE2 production in the absence of an effect on COX-2. Conversely, the enhancement of COX-2 expression was, in basal conditions, the only effect of DETA/NO related to PGE2 increase. In the case of LPS-stimulated cultures the opposite effects on precursor release and COX-2 expression resulted in an unmodified levels of PGE2. Thus, COX-2 expression appears to be the main target for the action of low and prolonged production of NO (as for DETA/NO), whereas short-lasting and high levels of NO seem to target the availability of AA. Our analysis does not allow, however to appreciate an additional effect of the two NONOates on COX activity, as proposed in other studies.27 The mechanism by which NO modulates COX-2 expression and AA availability in our cell system is under investigation. We also noted that DETA/NO but not PAPA/NO caused a measurable decrease of neutral red uptake suggesting a possible effect of low and long lasting NO level on either viability or activation of RAW 264.7 cells. In conclusion, our data indicate that the complex effects of NO on important biosynthetic pathways of macrophage functions are depending on the level and time of exposure to NO as well as on the activation state of the cells. The availability of controlled sources of NO is therefore important in dissecting the molecular targets and mechanisms affected by this potent and multi-faceted free radical.
Effect of nitric oxide on PGE2 and TNF- in RAW 264.7 cell / 179
MATERIALS AND METHODS Reagents RPMI 1640 medium without glutamine was from HyClone (Cramlington, UK), Glutamax I supplementcontaining L-alanyl-glutamine was from Life Technologies (San Giuliano Milanese, Italy). LPS (serotype 026:B6), Antibodies to PGE2 and PGE2 were obtained from Sigma (Milan, Italy). NG-monomethyl-L-arginine (NMMA), was from Cayman (Ann Arbor, MI, USA), PAPA/NO and DETA/NO were obtained from Alexis (Laufenlfingen, Switzerland). [3H]AA (60-100 Ci/mmol) and [3H]Prostaglandin E2 (PGE2) (171 Ci/mmol) were from Du Pont Nuclear (New England).
Cell culture The mouse monocyte/macrophage cell line RAW 264.7 was maintained in RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 10% heatinactivated fetal calf serum, 2 mM of Glutamax I (complete RPMI 1640 medium). All experiments were performed using complete RPMI. RAW 264.7 cells were plated at a density of 6105 cells/cm2. Cell cultures were allowed to adhere overnight, and stimulated with the indicated inducers or inhibitors after a medium change. Then the cell supernatants were removed, centrifuged, and stored at 20C, until tested.
PGE2 measurement and western blot analysis The level of PGE2 released into culture media was quantified using a specific radioimmunoassay as previously described.22 Detection limit was 25 pg/ml. For the evaluation of COX-2 expression, RAW 264.7 proteins were obtained from cell lysates as previously described22 and quantified by BCA assay protein (Pierce, Illinois). Equal amounts of proteins were subjected to western blot analysis using a specific antibody against COX-2, which does not cross react with COX-1.23 The Amersham ECL system was used to detect the primary antibodies. Purified COX-2 from sheep placenta was used as standard control. Determination of COX-2 expression level was performed using the GS-700 Imaging densitometer (Bio-Rad). The intensity of the bands was evaluated semiquantitatively and the variations in percentage were referred to the corresponding control bands.
Arachidonic acid release In order to estimate AA release, RAW 264.7 cells were incubated for 20–22 h in complete media supplemented with 0.5 Ci/ml [3H]AA, washed (3 times) with complete media, and incubated at 37C with the indicated test substances for 2 h and 5 h. The supernatants were then collected and counted for radioactivity. The incorporation pattern of [3H]AA in RAW 264.7 cells, after a 22 h labelling period, was: phosphatidylcholine, 16%; phosphatidylethanolamine, 37%; phosphatidylinositol, 22%; phosphatidylserine, 3%; neutral lipids, 7%. Such [3H]AA distribution remained identical after labelling periods of 40 h and 48 h.
Enzyme-linked immunosorbant assay (ELISA) Culture media from stimulated RAW cells were isolated centrifuged and stored at 70C until measured for TNF
using a commercially available ELISA (Cytoscreen immunoassay kit, Biosource International, Camarillo, CA, USA). After the addition of chromogen, the absorbance at 450 nm was measured in a micro plate reader (Model 550 Biorad).
Neutral red uptake Neutral red is a supravital stain, which is actively included by viable cells, but cannot be retained by non-viable cells.24 Neutral red is known to be accumulated in the lysosomes of macrophages and it is also used to identify and detect the activated state of these cells.25 Briefly, cells (1105 well) into 96-well plates in complete RPMI medium were washed and neutral red (50 g/ml) was added. The plates were incubated for 3 h and at the end of this incubation period cells were washed to remove excess dye and blotted dry. The incorporated dye was resuspended in ethanol (50%) containing glacial acetic acid (1%), and the absorbance measured at 550 nm in a micro plate reader (Model 550 Biorad). Results were calculated as percentage of neutral red incorporated by control cells.
REFERENCES 1. Wink DA, Mitchell JB (1998) Chemical biology of nitrix oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radical Biology and Medicine 25: 434–456. 2. Stamler JS (1994) Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 78:931–936. 3. Paludan SR (2000) Synergistic action of pro-inflammatory agents: cellular and molecular aspects. J Leukoc Biol 67:18–25. 4. Eigler A, Moeller J, Endres S (1995) Exogenous and endogenous nitric oxide attenuates Tumor Necrosis Factor synthesis in the murine macrophage cell line RAW 264.7. J Immunol 154:4048–4054. 5. Iuvone T, D’Acquisto F, Carnuccio R, Di Rosa M, (1996) Nitric oxide inhibits LPS-induced Tumor necrosis Factor synthesis in vitro and in vivo. Life Sci 59(13):207–211. 6. Lander HM, Sehajpal P, Levine DM, Novogrodsky A (1993) Activation of human peripheral blood mononuclear cells by nitric oxide-generating compounds. J Immunol 150:1509–1516. 7. Herschman HR (1996) Prostaglandin synthase 2. Biochim Biophys Acta 1299:125–140. 8. Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie MG (1993) Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci 90:7240–7244. 9. Guastadisegni C, Minghetti L, Nicolini A, Polazzi E, Ade P, Balduzzi M, Levi G (1997) Prostaglandin E2 synthesis is differentially affected by reactive nitrogen intermediates in cultured rat microglia and RAW 264.7 cells. FEBS letters 413:314–318. 10. Kanner J, Harel S, Granit R (1992) Nitric oxide, an inhibitor of lipid oxidation by lipoxygenase, cyclooxygenase and hemoglobin. Lipids 27:46–49. 11. Tsai AL, Wei C, Kulmacz RJ (1994) Interaction between nitric oxide and prostaglandin H synthase. Arch Biochem Biophys 313:367–372. 12. Minghetti L, Polazzi E, Nicolini A, Creminon C, Levi G (1996) Interferon-gamma and nitric oxide down-regulate lipopolysaccharide-induced prostanoid production in cultured rat microglial cells by inhibiting cyclooxygenase-2 expression. J Neurochem 66:1963–1970. 13. Maragos CM, Wang JM, Hrabie JA, Oppenheim JJ, Keefer LK (1993) Nitric oxide/nucleophile complexes inhibit the in vitro proliferation of A375 melanoma cells via nitric oxide release. Cancer Res 53:564–568.
180 / Guastadisegni et al. 14. Maragos CM, Morley D, Wink DA, Dunams TM, Saavedra JE, Hoffman A, Bove AA, Isaac L, Hrabie JA, Keefer LK (1991) Complexes of NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects. J Med Chem 34:3242–3247. 15. Wink DA, Cook JA, Krishna MC, Hanbauer I, DeGraff W, Gamson J, Mitchell JB (1995) Nitric oxide protects against alkyl peroxide-mediated cytotoxicity: further insights into the role nitric oxide plays in oxidative stress. Arch Biochem Biophys 319:402–407. 16. Mesmer UK, Brune B (1996) Nitric oxide (NO) in apoptotic versus necrotic RAW 264.7 macrophage cell death: the role of NO-donor exposure, NAD + content, and p53 accumulation. Arch Biochem Biophys 327:1–10. 17. Stamler JS, Singel DJ, Loascalzo J (1992) Biochemistry of nitric oxide and its redox-activated forms. Science 258:1898–1902. 18. Deakin AM, Payne AN, Whittle BJR, Moncada S (1995) The modulation of IL6 and TNF- release by nitric oxide following stimulation of J774 cells with LPS and IFN-. Cytokine 7(5): 408–416. 19. Peng HB, Libby P, Liao JK (1995) Induction and stabilization of I kappa B alpha by nitric oxide mediates inhibition of NF-kappa B. J Biol Chem 270(23):14214–14219.
CYTOKINE, Vol. 19, No. 4 (21 August, 2002: 175–180) 20. Wang S, Wang W, Wesley RA, Danner RL (1999) A Sp1 binding site of the Tumor Necrosis Factor promoter functions as a nitric oxide response element. J Biol Chem 274(47):33190–33193. 21. Goodwin DC, Landino LM, Marnett LJ (1999) Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis. FASEB J 13:1121–1136. 22. Minghetti L, Levi G (1995) Induction of prostanoid biosynthesis by bacterial lipopolysaccharide and isoproterenol in rat microglial cultures. J Neurochem 65:2690–2698. 23. Habib A, Cre`minon C, Frobert Y, Grassi J, Pradelles P, Maclouf J (1993) Demonstration of an inducible cyclooxygenase in human endothelial cells using antibodies raised against the carboxylterminal region of the cyclooxygenase-2. J Biol Chem 268(31): 23448–23454. 24. Filman DJ, Brawn RJ, Dandliker WB (1975) Intracellular supravital stain delocalization as an assay for antibody-dependent complement-mediated cell damage. J Immunol Methods 6:189–207. 25. Sipka S, Antal-Szalmas P, Szollosi I, Lakos G, Szegedi G (2000) Ceramide stimulates the uptake of neutral red in human neutrophils, monocytes, and lymphocytes. Ann Hematol 79(2): 83–85.
MODULATION OF PGE2 AND TNF BY NITRIC OXIDE IN RESTING AND LPS-ACTIVATED RAW 264.7 CELLS Cecilia Guastadisegni,1 Alessia Nicolini,2 Maria Balduzzi,3 Maria Antonietta Ajmone-Cat,2 Luisa Minghetti2 Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNF play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNF synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNF release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE2 production, while the LPS-stimulated production was slightly increased only by PAPA/ NO. The modulation of PGE2 synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules. 2002 Elsevier Science Ltd. All rights reserved.
Macrophages play a major role in host defence against infection and cancer. Activation of macrophages by lipopolysaccharide (LPS) enhances the production and release of inflammatory mediators, including cytokines, arachidonic acid metabolites and From the 1Laboratory of Environmental Hygiene, 2Neurobiology Section, Laboratory of Pathophysiology, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome and 3Toxicology Biochemical Science Section, ENEA, Via Anguillarese 301, Rome, Italy Correspondence to: Cecilia Guastadisegni, Laboratorio di Igiene Ambientale, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Roma, Italy. Fax: + +39-6-49387083; Tel: + +39-649902885; E-mail: [email protected] Received 6 October 2001; received in revised form 8 June 2002; accepted for publication 19 June 2002 1043–4666/02/$-see front matter 2002 Elsevier Science Ltd. All rights reserved. KEY WORDS: nitric oxide/nitric oxide-releasing compounds/ prostaglandins/Tumor Necrosis Factor/cyclooxygenase/arachidonic acid CYTOKINE, Vol. 19, No. 4 (21 August), 2002: pp 175–180
nitric oxide (NO). Nitric oxide has been shown to be involved in numerous biological effects, both physiological and pathological, and to exert several regulatory functions. The effects exerted by NO may be either direct or mediated by the reactive nitrogen oxide species (RNOS) formed in an aerobic biological environment. Indeed, NO react with oxygen, superoxide and transition metals, generating NOx, peroxynitrite and heme complexes, respectively.1 The products of these reactions can in turn support additional nitrosative reactions with nucleophilic cellular molecules.2 Among the several macrophage functions that might be regulated by NO are the syntheses of Tumor Necrosis Factor (TNF) and prostaglandins (PGs), which are important mediators of inflammation and cellular immune response. TNF, originally defined by its antitumoral activity, is now recognized as a potent mediator of 175
176 / Guastadisegni et al.
inflammatory responses possessing antibacterial and antiviral properties.3 Positive and negative regulation of TNF synthesis by NO has been reported, depending on the NO donor used.4–6 Prostaglandins, and in particular PGE2, is regarded as a potent pro-inflammatory molecule, however, increasing evidence indicated that they can also exert anti-inflammatory functions which are important for the resolution of the inflammatory response.7 They are metabolites of arachidonic acid (AA) through the cyclooxygenase (COX) metabolic pathway. As for NOS, COX exists in a constitutive isoform, which is expressed in most tissue and is involved at the maintenance of several physiological functions, and in an inducible isoform (COX-2), which is the major isoform expressed in activated inflammatory cells. Since COX is a heme protein, it has been proposed that NO enhances COX activity by interacting with the heme group, as described for the enzyme guanylate cyclase, one of the major target of NO.8 However, both positive and negative regulation of PG synthesis by NO have been reported.8–11 As suggested by Wink and Mitchell,1 one possible explanation of these conflicting results is that NO might affect COX activity by other mechanisms, in alternative to the interaction with the metal centers of the enzyme. In line with this view, we have previously demonstrated that NO modulates prostanoid synthesis by acting at different steps in the PGs synthetic pathway, including AA release and expression of COX-2.9,12 The apparent conflicting results reported in the literature on NO regulatory activities may also be related to the different experimental conditions and in particular to the specific source of NO used. Recently a group of compounds, NONOates has been introduced, which do not require enzymatic metabolism to release NO and release predictable amounts of NO at a chemically defined rate, allowing well-controlled biological studies.13,14 In the present study, we have used PAPA/NO, with a half-life of 15 min, and DETA/NO, characterised by a half-life of 20 h, to expose the cell cultures either to high levels of NO for brief time (PAPA/NO) or to low levels of NO for long time (DETA/NO). By this experimental approach we have found that level and time of exposure to NO are both crucial in determining the molecular target as well as the final result of the interactions between NO and inflammatory molecules.
RESULTS Effects of NONOates on TNF synthesis NONOates are nucleophilic complexes of NO, spontaneously releasing approximately 2 moles of NO per molecule of starting material after addition to
CYTOKINE, Vol. 19, No. 4 (21 August, 2002: 175–180)
Figure 1. Effect of PAPA/NO and DETA/NO on basal TNF production in RAW 264.7 cells. The cells were incubated in the presence of the NOS inhibitor NMMA (400 M) with or without DETA/NO (deta) and PAPA/NO (papa) at two different concentrations 0.5 and 1 mM for 5 h, 16 h and 24 h. Values are meansS.E.M. of three independent experiments assayed in duplicate. *P<0.025, **P<0.005, ***P<0.001, ****P<0.0005 compared with the corresponding NMMA treated cells.
aqueous solutions at neutral pH. Given the half-lives of PAPA/NO (t Y 15 min) and DETA/NO (t Y 20 h), the addition to the culture media of the same amount of these two NONOates results in exposure of cells to different levels of NO. Indeed, 1 mM PAPA/NO generates an immediate peak of 22 M NO, that after 1 h declines to around 1–2 M.15 On the other hand, DETA/NO, with the predicted half life of 20 h, releases a much lower but long lasting NO output.16 Cell cultures were incubated for 24 h with individual NONOates without or with LPS. In order to avoid interference with the endogenously produced NO, the effects of NONOates were studied in the presence of the NO-synthase inhibitor NG-monomethyl-L-arginine (NMMA) (400 M), able to inhibit by over 70% NO production in RAW cells.9 At the end of the incubation time, supernatants were collected and analysed for TNF contents. RAW 264.7 cells spontaneously released detectable levels of TNF after 5 h of incubation (8416 pg/ ml n=3), which further increased at 16 h of incubation (23454 pg/ml n=3) and remained stable at 24 h (21856 pg/ml n=3). The presence of 1 g/ml of LPS remarkably increased the release of TNF, which reached maximal levels between 5 h and 16 h of incubation (179324 pg/ml and 2037118 pg/ml, after 5 h and 16 h respectively, n=3). In basal conditions, PAPA/NO and DETA/NO, at two different concentrations (0.5 and 1 mM) had opposite effects on TNF release. Indeed, we found that DETA/NO decreased the basal TNF release at 16 h and 24 h, whereas PAPA/NO caused an increase in the synthesis of TNF, already detectable after 5 h of incubation with the higher concentration of the
Effect of nitric oxide on PGE2 and TNF- in RAW 264.7 cell / 177
Figure 2. Effect of PAPA/NO and DETA/NO on basal and LPS induced PGE2 production in RAW 264.7 cells. Cells were incubated with or without 1 g/ml LPS in the presence of the NOS inhibitor NMMA (400 M) with or without DETA/NO (deta) and PAPA/NO (papa) at two different concentrations 0.5 and 1 mM for 24 h. One experiment assayed in triplicate representative of four is shown.
NONOate (Fig. 1). Conversely, PAPA/NO did not affect LPS-induced TNF release at any concentration and at any experimental time point, while DETA/NO slightly reduced the stimulation of TNF at 16 h (data not shown).
Effects of NONOates on PGE2 synthesis The effect of the two NO-donors on PG synthesis was evaluated in the same experimental conditions described above. In Figure 2, one representative experiment of four is shown. Both NONOates dosedependently increased basal PGE2 production. DETA/NO increased PGE2 by 19460% and 23464% (n=4) of control levels at 0.5 mM and 1 mM respectively. Similarly, PGE2 production in the presence of PAPA/NO was 17417% and 23868% (n=4) at 0.5 mM and 1 mM, respectively. The presence of 1 g/ml of LPS induced a significant increase in PGE2 accumulation (287 pg/ml and 572380 pg/ml, in basal and LPS-stimulated RAW 264.7, respectively, n=4), consistently with previous studies.9 LPS-stimulated PGE2 production was increased in the presence of PAPA/NO, while DETA/NO had no effect on this production. The presence of PAPA/NO consistently increased the LPSinduced PGE2 synthesis of 13413% (P<0.05, n=4) at 0.5 mM and 15311% (P<0.005, n=4) at 1 mM concentration.
Effects of NONOates on AA release and COX-2 expression To investigate the possible effect of the two compounds on the release of PGs precursor AA, cells were prelabelled with [3H]AA and then incubated for 2 h
and 5 h with PAPA/NO and DETA/NO, without or with 1 g/ml of LPS. As previously described the two NONOates affected in a different way the [3H]AA release in RAW 264.7 cells (Table 1). PAPA/NO (t Y 15 min) increased both basal and LPS-induced [3H]AA release after 5 h of incubation, whereas after 2 h it was ineffective on unstimulated cells, and decreased the AA-release from LPS-stimulated cells. At variance, DETA/NO (t Y 20 h) showed only a transient increase after 2 h in the presence of LPS (Table 1). We then analysed the expression of COX-2 in unstimulated and LPS-stimulated RAW 264.7 cells, in the presence of the two NONOates (Fig.3). As previously showed,9 COX-2 expression was clearly detectable in unstimulated RAW 264.7 cells, which was further enhanced by the exposure to LPS (1 g/ml). When RAW 264.7 cells were incubated for 24 h with PAPA/NO or DETA/NO, COX-2 expression was moderately increased. On the other hand, DETA/NO showed a much stronger ability to increase COX-2 expression in LPS-stimulated cells than PAPA/NO (Fig. 3).
Cell viability Incubation of RAW264.7 cells with PAPA/NO either with or without LPS had little or no effect on cell viability as measured by neutral red uptake at 24 h. However incubation with DETA/NO at 0.5 mM and 1 mM significantly reduced neutral red uptake and cellular viability or activation at 24 h (6610%, n=3 and 5411%, n=3 P<0.05; respectively). Concurrent incubation of cells with DETA/NO and LPS did not result in any greater decrease in neutral red uptake as compared to DETA/NO only (not shown).
DISCUSSION Cytotoxic and cytoprotective actions of NO have been reported. This paradox lies on NO reactivity and on its ability to react directly or indirectly with several active groups.17 With the use of selected NO donors, characterised by different half lives and kinetics of NO release, we showed that level and time of exposure are crucial in determining the effect of NO on PGE2 and TNF macrophage synthesis. Our results indicate that TNF is downregulated in RAW 264.7 cells exposed to low level of NO for long time, while is augmented when cells are exposed to high level of NO for short time. Previous studies on RAW 264.7 and J774 macrophages exposed to SIN-1, a donor of the two radicals O2and NO, which in turn combine to form peroxynitrite,2 showed a decrease in the synthesis of TNF compared to control cultures.4,5 As formation of peroxynitrite within the cells is likely to occur in the presence of low concentration
178 / Guastadisegni et al.
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TABLE 1. Effect of PAPA/NO and DETA/NO on basal and LPS-induced [3H]AA release in RAW 264.7 cells. % [3H]AA release 2h
Control PAPA/NO 0.5 mM PAPA/NO 1 mM DETA/NO 0.5 mM DETA/NO 1 mM
5h
Control
LPS 1 g/ml
Control
LPS 1 g/ml
100 1043.2 1014.1 1045.9 11610
1307.2 813.1 813.0 14015.4 15510.6*
100 1204.8* 1346.7* 1033.8 998.1
1265.8 1476.7* 1729.5* 945.6 985.5
Cells were prelabelled with [3H]AA and then incubated for 2 h and 5 h with PAPA/NO and DETA/NO (0.5 and 1 mM) with or without 1 g/ml of LPS, in the presence of NMMA. The radioactivity released by untreated cells was taken as 100%. *P<0.05 compared with the corresponding control.
of NO,1 the effect of DETA/NO, responsible for low and prolonged NO release may be mediated by peroxynitrite. On the other hand, other studies on J774 cells showed an increased TNF synthesis upon exposure to NO, using S-nitroso-N-acetylpenicillamine (SNAP) as NO donor,18 consistently with our data in the presence of PAPA/NO. The mechanism by which NO results in these multiple effects is not known. NO can bind superoxide anion with extremely high affinity and submicromolar amounts of NO, like in the presence of DETA/NO, are likely to bind superoxide.1,19 NO has been shown to activate the TNF promoter trough the binding in Sp1 site.20 This region is therefore NO responsive and it may not respond to peroxynitrite, this may explain the difference between the two NONOates. Indeed, we
Figure 3. Effect of PAPA/NO and DETA/NO on basal and LPSstimulated COX-2 expression in RAW 264.7 cells. Cells were incubated for 24 h in complete medium, without (panel A) or with (panel B) 1 g/ml LPS in the presence of NMMA and with DETA/NO (deta) and PAPA/NO (papa) at two different concentrations 0.5 and 1 mM. Cell lysates were prepared and equal amounts of protein (25 g) were analysed by western blot using anti COX-2 specific antibodies (1:500). Protein bands were visualised using horseradish peroxidase-conjugated secondary antibodies and ECL. One experiment representative of three is shown.
showed that the two NONOates have similar effects on PGE2 synthesis, which was increased in unstimulated cultures and unaffected in LPS-stimulated cultures. However, the activities of the two NONOates were the results of distinct mechanisms. The enhancement of basal PGE2 production observed in RAW 264.7 cells after exposure to PAPA/NO seemed related to both an increased availability of the PGE2 precursor AA, and to a slight increase in COX-2 expression. In the LPSstimulated cultures the effect on AA was not sufficient to enhance PGE2 production in the absence of an effect on COX-2. Conversely, the enhancement of COX-2 expression was, in basal conditions, the only effect of DETA/NO related to PGE2 increase. In the case of LPS-stimulated cultures the opposite effects on precursor release and COX-2 expression resulted in an unmodified levels of PGE2. Thus, COX-2 expression appears to be the main target for the action of low and prolonged production of NO (as for DETA/NO), whereas short-lasting and high levels of NO seem to target the availability of AA. Our analysis does not allow, however to appreciate an additional effect of the two NONOates on COX activity, as proposed in other studies.27 The mechanism by which NO modulates COX-2 expression and AA availability in our cell system is under investigation. We also noted that DETA/NO but not PAPA/NO caused a measurable decrease of neutral red uptake suggesting a possible effect of low and long lasting NO level on either viability or activation of RAW 264.7 cells. In conclusion, our data indicate that the complex effects of NO on important biosynthetic pathways of macrophage functions are depending on the level and time of exposure to NO as well as on the activation state of the cells. The availability of controlled sources of NO is therefore important in dissecting the molecular targets and mechanisms affected by this potent and multi-faceted free radical.
Effect of nitric oxide on PGE2 and TNF- in RAW 264.7 cell / 179
MATERIALS AND METHODS Reagents RPMI 1640 medium without glutamine was from HyClone (Cramlington, UK), Glutamax I supplementcontaining L-alanyl-glutamine was from Life Technologies (San Giuliano Milanese, Italy). LPS (serotype 026:B6), Antibodies to PGE2 and PGE2 were obtained from Sigma (Milan, Italy). NG-monomethyl-L-arginine (NMMA), was from Cayman (Ann Arbor, MI, USA), PAPA/NO and DETA/NO were obtained from Alexis (Laufenlfingen, Switzerland). [3H]AA (60-100 Ci/mmol) and [3H]Prostaglandin E2 (PGE2) (171 Ci/mmol) were from Du Pont Nuclear (New England).
Cell culture The mouse monocyte/macrophage cell line RAW 264.7 was maintained in RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, 10% heatinactivated fetal calf serum, 2 mM of Glutamax I (complete RPMI 1640 medium). All experiments were performed using complete RPMI. RAW 264.7 cells were plated at a density of 6105 cells/cm2. Cell cultures were allowed to adhere overnight, and stimulated with the indicated inducers or inhibitors after a medium change. Then the cell supernatants were removed, centrifuged, and stored at 20C, until tested.
PGE2 measurement and western blot analysis The level of PGE2 released into culture media was quantified using a specific radioimmunoassay as previously described.22 Detection limit was 25 pg/ml. For the evaluation of COX-2 expression, RAW 264.7 proteins were obtained from cell lysates as previously described22 and quantified by BCA assay protein (Pierce, Illinois). Equal amounts of proteins were subjected to western blot analysis using a specific antibody against COX-2, which does not cross react with COX-1.23 The Amersham ECL system was used to detect the primary antibodies. Purified COX-2 from sheep placenta was used as standard control. Determination of COX-2 expression level was performed using the GS-700 Imaging densitometer (Bio-Rad). The intensity of the bands was evaluated semiquantitatively and the variations in percentage were referred to the corresponding control bands.
Arachidonic acid release In order to estimate AA release, RAW 264.7 cells were incubated for 20–22 h in complete media supplemented with 0.5 Ci/ml [3H]AA, washed (3 times) with complete media, and incubated at 37C with the indicated test substances for 2 h and 5 h. The supernatants were then collected and counted for radioactivity. The incorporation pattern of [3H]AA in RAW 264.7 cells, after a 22 h labelling period, was: phosphatidylcholine, 16%; phosphatidylethanolamine, 37%; phosphatidylinositol, 22%; phosphatidylserine, 3%; neutral lipids, 7%. Such [3H]AA distribution remained identical after labelling periods of 40 h and 48 h.
Enzyme-linked immunosorbant assay (ELISA) Culture media from stimulated RAW cells were isolated centrifuged and stored at 70C until measured for TNF
using a commercially available ELISA (Cytoscreen immunoassay kit, Biosource International, Camarillo, CA, USA). After the addition of chromogen, the absorbance at 450 nm was measured in a micro plate reader (Model 550 Biorad).
Neutral red uptake Neutral red is a supravital stain, which is actively included by viable cells, but cannot be retained by non-viable cells.24 Neutral red is known to be accumulated in the lysosomes of macrophages and it is also used to identify and detect the activated state of these cells.25 Briefly, cells (1105 well) into 96-well plates in complete RPMI medium were washed and neutral red (50 g/ml) was added. The plates were incubated for 3 h and at the end of this incubation period cells were washed to remove excess dye and blotted dry. The incorporated dye was resuspended in ethanol (50%) containing glacial acetic acid (1%), and the absorbance measured at 550 nm in a micro plate reader (Model 550 Biorad). Results were calculated as percentage of neutral red incorporated by control cells.
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