Inhibition of Inducible Nitric Oxide Synthase by Peroxisome Proliferator-Activated Receptor Agonists: Correlation with Induction of Heme Oxygenase 1 This information is current as of September 24, 2021. Paul R. Colville-Nash, Saima S. Qureshi, Dean Willis and Derek A. Willoughby J Immunol 1998; 161:978-984; ; http://www.jimmunol.org/content/161/2/978 Downloaded from References This article cites 37 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/161/2/978.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Inhibition of Inducible Nitric Oxide Synthase by Peroxisome Proliferator-Activated Receptor Agonists: Correlation with Induction of Heme Oxygenase 11 Paul R. Colville-Nash, Saima S. Qureshi, Dean Willis,2 and Derek A. Willoughby Genetic knock-out in mice of peroxisome proliferator-activated receptor-a (PPARa) can prolong inflammation in response to leukotriene B4. Although cyclooxygenase 2 has been shown to be induced by PPAR activation, the effect of PPAR agonists on the key inflammatory enzyme systems of nitric oxide synthase (NOS) and stress proteins has not been investigated. The effect on these a of naturally occurring eicosanoid PPAR agonists (leukotriene B4 and 8(S)-hydroxyeicosatetraenoic acid, which are PPAR se- D12 g a lective; PGA2, PGD2, PGJ2, and PGJ2, which are PPAR selective) and the synthetic PPAR agonist Wy14,643 was examined Downloaded from in activated RAW264.7 murine macrophages. Leukotriene B4 and 8(S)-hydroxyeicosatetraenoic acid stimulated nitrite accumu- D12 lation, indicative of enhanced NOS activity. PGA2, PGD2, PGJ2, PGJ2, and Wy14,643 reduced nitrite accumulation, with D12 PGJ2 being the most effective. The mechanism behind this reduction was examined using Western blotting. Inhibition of nitrite accumulation was associated with a fall in inducible NOS protein and an induction of heme oxygenase 1, correlating both dose dependently and temporally. Other proteins examined (cyclooxygenase 2, heme oxygenase 2, heat shock protein 70, and glucose- regulated protein 78) were unaffected. The data suggest that naturally occurring PPAR agonists can inhibit the inducible NOS enzyme pathway. This inhibition may be mediated by modulation of the stress protein, heme oxygenase 1. Thus, the generation http://www.jimmunol.org/ of eicosanoid breakdown products during inflammation may contribute to its eventual resolution by activation of the PPAR system. This system may thus represent a novel target for therapeutic intervention in inflammatory disease. The Journal of Immunology, 1998, 161: 978–984. he inflammatory response involves the sequential activa- One subclass of PGs, the cyclopentenone PGs, has been shown tion of a number of signaling pathways, resulting in the to cause a variety of biologic effects, including cessation of cell T production of a variety of mediators that act both locally growth (5) and induction of cell differentiation (6). This has im- and systemically. Since the demonstration that part of the anti- plications for a variety of diseases, such as tumorigenesis and de- by guest on September 24, 2021 inflammatory activity of aspirin stems from inhibition of cycloox- fects in lipid physiology. In the latter field, it has been demon- ygenase (COX)3 (1) and a reduction in the formation of PG, there strated that the cyclopentenone PGs are among the endogenous have been enormous advances in the understanding of this path- activators of a class of nuclear receptors, the peroxisome prolif- way, as well as the discovery of a role for a variety of other path- erator-activated receptors (PPARs; for a review, see Refs. 7 and 8). ways in inflammation, such as nitric oxide synthase (NOS; partic- These were originally discovered from work with early hypolipi- ularly the inducible isoform (iNOS) (2)) and nitric oxide (NO), the demic drugs such as the fibrates. Ligands for these receptors in- stress proteins (heat shock proteins (HSPs)) (3), and the heme ox- clude a variety of other drugs such as the antidiabetic thiazo- ygenase (HO) (4) pathway. Much interest is now focused on the lidinediones, xenobiotics, certain members of the eicosanoid interactions between these systems, their potential role in modu- pathways such as the leukotrienes (LT), and fatty acids. Three lating inflammation, and their role as therapeutic targets for inter- subtypes have been described to date, PPARa, PPARb (also re- vention in inflammation and other forms of disease. lated to PPARd, murine fatty acid-activated receptor, and NUC1) and PPARg. Their activation leads to the induction of a variety of genes such as those coding for the enzymes for b- and v-oxidation Department of Experimental Pathology, St. Bartholomew’s and The Royal London of fatty acids (9), transferrin (10), and transthyretin (11). In addi- School of Medicine and Dentistry, London, United Kingdom tion to this information, it is known that cyclopentenone PGs are Received for publication November 12, 1997. Accepted for publication March capable of modifying the stress response by activation of heat 23, 1998. shock factor 1 (HSF-1 (5)) and induction of HSP70 (12) and HO-1 The costs of publication of this article were defrayed in part by the payment of page (13), which have been implicated in the biologic actions of this charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. class of PGs. Whether this is mediated via the PPARs is at present 1 P.R.C.-N. was the recipient of a fellowship sponsored by the Hyal Research Foun- unclear, but it has been demonstrated that PPARs are associated dation, Canada. D.W. was supported by ONO Pharmaceutical Co. Ltd., Japan. with HSP72 (14), and it has been hypothesized that this association 2 Address correspondence and reprint requests to Dr D. Willis, Department of Ex- may be important in the signaling pathways of these receptors. perimental Pathology, St. Bartholomew’s and The Royal London School of Medicine Recently, it has been demonstrated that the inflammatory re- and Dentistry, Charterhouse Square, London, EC1 M 6BQ, U.K. E-mail address: [email protected] sponse to leukotriene B4 (LTB4) is prolonged in genetically modified mice with homozygous knock-out of PPARa (15). 3 Abbreviations used in this paper: COX, cyclooxygenase; NOS, nitric oxide syn- thase; iNOS, inducible nitric oxide synthase; NO, nitric oxide; HSP, heat shock pro- Devchand et al. (15) proposed that, in wild-type mice, LTB4 teins; HO, heme oxygenase; PPAR, peroxisome proliferator-activated receptor; HSF, would activate PPARa, induce fatty acid oxidation pathways, heat shock factor; LTB4, leukotriene B4; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-di- phenyl-tetrazolium; GRP78, glucose-regulated protein 78; 8(S)-HETE, 8 hydroxy- which in turn would degrade lipid mediators of inflammation, 5,9,11,14-eicosatetraenoic acid; ZnDPP, zinc deuteroprotoporphyrin. and concluded that activators of PPARa should be Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 979 anti-inflammatory. However, this enhanced degradation of lipid U.K.). Anti-rabbit IgG for ECL (Amersham) was used at dilutions of mediators has not been demonstrated in this model, and a va- 1:2000 for HO-1, HO-2, and GRP78, 1:3000 for COX-2, and 1:4000 for riety of other inflammatory mechanisms such as those suggested iNOS and HSP70. Bands were recorded on x-ray film (Fuji RX, Genetic Research Instrumentation, Dunmow, U.K.) for permanent record. Repre- above may also be important in the effects attributable to PPAR sentative blots from three experiments are presented. activation. To date, the effect of cyclopentenone PGs and other PPAR ago- Statistical analysis nists on iNOS in macrophages, a key cell and enzyme system in Results are representative of three experiments with n 5 6 in each group. inflammation and other pathologies, has not been described. In this Significance of data was examined by analysis of variance (ANOVA) fol- D12 g lowed by a post hoc Bonferroni-corrected t test (Instat, GraphPad Software, study, we demonstrate that PGJ2, a PPAR agonist, is a potent inhibitor of iNOS activity and protein expression in stimulated University of Amsterdam, The Netherlands). RAW264.7 macrophages. Protein expression and inhibitor studies Results implicate activation of HO-1 in this phenomenon. Effects of various PPAR agonists on viability and iNOS activity Materials and Methods in stimulated RAW264.7 macrophages Culture and stimulation of murine RAW264.7 macrophages Stimulation of macrophages with IFN-g and LPS routinely in- creased nitrite levels in medium by .12-fold after 18 h, compared RAW264.7 macrophages (European Collection of Animal Cell Cultures) were cultured as described previously (16). Cells were passaged and grown with nonstimulated cultures (data not shown). Viability of cell cul- m to 90% confluence in 96-well plates (Greiner) for analysis of nitrite pro- tures (Fig. 1) treated with up to 10 g/ml of LTB4,8(S)-hydroxy- duction and viability or in 75-cm2 culture flasks (Greiner, Stonehouse, Downloaded from eicosatetraenoic acid (8(S)-HETE), PGA2, PGD2, and PGJ2 was U.K.) for Western blot analysis of protein expression.