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

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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-␣ (PPAR␣) 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 ␣ of naturally occurring eicosanoid PPAR agonists (leukotriene B4 and 8(S)-hydroxyeicosatetraenoic acid, which are PPAR se- ⌬12 ␥ ␣ 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- ⌬12 lation, indicative of enhanced NOS activity. PGA2, PGD2, PGJ2, PGJ2, and Wy14,643 reduced nitrite accumulation, with ⌬12 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, PPAR␣, PPAR␤ (also re- vention in inflammation and other forms of disease. lated to PPAR␦, murine fatty acid-activated receptor, and NUC1) and PPAR␥. Their activation leads to the induction of a variety of genes such as those coding for the enzymes for ␤- and ␻-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 PPAR␣ (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 PPAR␣, 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 PPAR␣ 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 ϭ 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- ⌬12 ␥ 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-␥ and LPS routinely in- creased nitrite levels in medium by Ͼ12-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- ␮ 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. Macrophages were not affected. However, Wy14,643 was found to be toxic at a con- stimulated for 18 h (unless otherwise indicated) by addition of 100 U/ml ␮ murine rIFN-␥ (Sigma Chemical, Poole, U.K.) and 0.1 ␮g/ml LPS (LPS, centration of 100 g/ml, although no effect on viability was ob- ␮ ⌬12 Escherichia coli serotype 0111:B4; Sigma Chemical). Concurrently ad- served at 30 g/ml or below. PGJ2 reduced viability at 10 ministered PPAR agonist compounds were dissolved in methyl acetate ␮g/ml. Only data derived from nontoxic doses of compounds was such that the final concentration of solvent in medium was 0.1% by vol- considered, therefore, in subsequent analysis of the results.

ume, with solvent alone as control. At this concentration, the solvent has no http://www.jimmunol.org/ LTB4 and 8(S)-HETE significantly increased nitrite accumula- effect on cellular functions examined in this study (data not shown). Ei- ␮ cosanoids were purchased from Biomol (Plymouth Meeting, PA), and tion in culture medium at higher dosages of 3 g/ml (38% rise) Wy14,643 was purchased from Caymen Chemical (Ann Arbor, MI). Zinc and 10 ␮g/ml (36%), respectively (Fig. 1). In contrast, Wy14,643 deuteroprotoporphyrin (ZnDPP; Porphyrin Products, Logan, UT) was ini- dose dependently reduced nitrite accumulation, causing a signifi- ϫ tially dissolved at 100 the required concentration in 0.1 M sodium hy- cant 60% decrease at the highest nontoxic dose of 30 ␮g/ml. droxide before dilution with appropriate media. At doses shown to be nontoxic, all PGs tested proved capable of Nitrite assay inhibiting iNOS activity at the higher doses investigated, indicated Nitrite accumulation in culture medium was used as a measure of cellular by a reduction in nitrite accumulation in test medium (Fig. 2). PGA at doses up to 1 ␮g/ml significantly increased nitrite pro- NO synthesis (17). This was assessed spectrophotometrically by addition 2 by guest on September 24, 2021 of 100 ␮l of Griess reagent (1% sulfanilamide and 0.1% N-(1-naphthyl- duction. Above this level, nitrite production was reduced, with a )ethylenediamine in 5% o-phosphoric acid; Sigma Chemical) to an equal ␮ 94% reduction at 10 g/ml. PGD2 did not elevate nitrite accumu- volume of sample medium in 96-well plates. Optical densities were mea- lation at low doses, but dose dependently reduced nitrite accumu- sured by dual wavelength analysis (OD 570–630 nm) in a plate reader ␮ (Biotek EL-310; Biotek Instruments, Winooski, VT); sodium nitrite was lation. This reached significance at 1 g/ml, and nitrite levels fell ␮ used as standard. by 51% at 10 g/ml. PGJ2 elicited a response similar to that of ␮ PGD2, although significance was not reached until 3 g/ml, with Cell viability assay ␮ ⌬12 a 69% fall at 10 g/ml. PGJ2 was the most potent inhibitor of Cell viability was assessed by measuring the ability of macrophages to nitrite accumulation, with reaching significance at 30 ng/ml, and reduce 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium (MTT) at the an 81% reduction at the highest nontoxic dose of 3 ␮g/ml. For end of experimental procedures (18). 5 mg/ml MTT (Sigma Chemical) was added to each well to give a final concentration of 0.5 mg/ml, and cells subsequent analysis of changes in protein expression in response to were incubated for 20 min, the medium removed, and the formazan product PPAR agonist treatment, the response to the most potent inhibitor ␮ ⌬12 dissolved in 100 l DMSO. Product formation was assessed spectropho- of iNOS, PGJ2, was investigated. tometrically by measuring the OD at a dual wavelength (OD 570–630 nm) in a plate reader (Biotek EL-310). ⌬12 Western blot analysis of the dose-dependent effects of PGJ2 Western blotting on stimulated RAW264.7 macrophages ␥ Test medium was removed from culture flasks and cells resuspended by Stimulation of macrophages with IFN- and LPS markedly in- cell scraper in 2 ml of fresh medium. Cells were pelleted by centrifugation creased iNOS protein expression at 18 h poststimulation (Fig. 3). ϫ ␮ ␮ ⌬12 (200 g, 4°C for 10 min), resuspended in 200 l of ice-cold protease At doses up to 0.3 g/ml, PGJ2 did not affect this level visibly. inhibitory buffer (1 mM PMSF, 1.5 mM pepstatin A, 0.2 mM leupeptin in Above this dosage, however, iNOS protein levels fell markedly, 0.05 M Tris buffer, pH 7.4; Sigma Chemical), and sonicated. Protein con- centrations of samples was determined by Bradford assay (Bio-Rad, Hemel with little expression visible, compared with stimulated control Hempstead, U.K.) and samples equilibrated to 2 mg/ml with protease in- cultures at the highest nontoxic dose of 3 ␮g/ml. These changes in hibitory buffer. An equal volume of Laemmli loading buffer was added, iNOS expression correlated with changes in the levels of HO-1 and samples were boiled for 5 min. Samples were loaded on 7.5% gels for present in these macrophages. Thus, at low doses, induction of SDS-PAGE for iNOS, glucose-regulated protein 78 (GRP78), HSP70, and HO-1 protein expression was evident, but increased markedly at COX-2 and 15% gels for HO-1 and HO-2. Proteins were electroeluted onto ⌬12 ␮ nitrocellulose membrane (Hybond C Super, 0.45 ␮m, Amersham, Little concentrations of PGJ2 of 0.3 g/ml and above. Additional Chalfont, U.K.). Protein expression was visualized by enhanced chemilu- lower m.w. bands seen at 3 ␮g/ml and 10 ␮g/ml are breakdown minescent detection (ECL; Amersham). The following polyclonal primary products of HO-1 (4). Abs and dilutions were used: rabbit anti-mouse iNOS, 1:5000 (Santa Cruz COX-2 protein levels were increased by stimulation of macro- Biotechnology, Santa Cruz, CA); rabbit anti-rat HO-1, 1:2000, rabbit anti- ␥ rat HO-2, 1:2000, and rabbit anti-rat GRP78, 1:2000 (Stressgen Biotech- phages with IFN- and LPS, but although minor variations in pro- nologies, Victoria, Canada); rabbit anti-mouse COX-2, 1:3000 (Caymen tein levels were observed, no consistent effect on protein expres- ⌬12 Chemical); rabbit anti-human HSP70, 1:4000 (Dako, High Wycombe, sion was seen in the presence of PGJ2 compared with 980 PPAR AGONISTS AND INHIBITION OF iNOS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 FIGURE 1. The effect of various eicosanoid and Wy14,643 PPAR li- FIGURE 2. The effect of various eicosanoid and Wy14,643 PPAR li- gands on cell viability in RAW264.7 murine macrophages. RAW264.7 gands on nitrite production in RAW264.7 murine macrophages. ␥ murine macrophages were stimulated with 100 U/ml IFN- and 100 ng/ml RAW264.7 murine macrophages were stimulated with 100 U/ml IFN-␥ LPS (controls) for 18 h in the presence of varying concentrations of LTB4, and 100 ng/ml LPS (controls) for 18 h in the presence of varying concen- ⌬12 8(S)-HETE, Wy14,643, PGA2, PGD2, PGJ2, and PGJ2. Cell viability trations of LTB ,8(S)-HETE, Wy14,643, PGA , PGD , PGJ , and Ͻ 4 2 2 2 was measured by MTT reduction assay. ***p 0.001 as measured by ⌬12PGJ . Nitrite production measured as accumulated product was as- ϭ 2 ANOVA with post hoc Bonferroni-corrected t test, n 6 in each group; sessed using the Griess reaction.*p Ͻ 0.05, **p Ͻ 0.01, ***p Ͻ 0.001 as representative data from three experiments. measured by ANOVA with post hoc Bonferroni-corrected t test, n ϭ 6in each group; representative data from three experiments.

stimulated control cultures, with no evidence of a dose-dependent effect (data not shown). Expression of HO-2, HSP70, and GRP78 In keeping with the results reported above, expression of COX-2 (Fig. 3) were also unaffected either by stimulation or by treatment was increased by stimulation with IFN-␥ and LPS, this being ev- ⌬12 with PGJ2. ident at 3 h and maintained through to 24 h. Variation in protein expression between control and treated cultures was seen, but no ␮ Western blot analysis of the time-dependent effects of 3 g/ml consistent pattern in the temporal changes was observed over the ⌬12 PGJ2 on stimulated RAW264.7 macrophages three experiments, and it was concluded that COX-2 expression ⌬12 Analysis of the time course of protein expression was conducted was unaffected by the presence of PGJ2 (data not shown). As ⌬12 ␮ using the highest nontoxic dose of PGJ2,3 g/ml. In non-PG- seen previously, neither stimulation nor the presence of the PG treated cultures, stimulation with IFN-␥ and LPS increased iNOS affected the expression of HO-2, HSP70, and GRP78 at any time expression (Fig. 4), this effect being evident at 6 h, greatest at 12 h, point examined (Fig. 4). and slightly reduced at 24 h. HO-1 was similarly induced (Fig. 4), with a longer delay before protein expression was enhanced, this The effect of ZnDPP on the inhibition of nitrite release by ⌬12 being evident at 12 h and most notable at 24 h. PGJ2 ⌬12 Treatment with PGJ2 caused a retardation in iNOS expres- Given the correlation between iNOS expression and HO-1 seen in sion, with increased expression first evident at 9 h poststimulation, the Western blot analysis, the effect of including a selective HO and the greatest response seen at 24 h (Fig. 4). This correlated with inhibitor, ZnDPP, was examined. As shown previously, addition of ⌬12 ␮ an earlier induction of HO-1, with much increased expression of PGJ2 at doses up to 3 g/ml was without effect on viability of this protein seen at 6 through 12 h poststimulation. This increased macrophages stimulated with IFN-␥ and LPS (Fig. 5). Addition of ⌬12 ␮ expression was lost at 24 h poststimulation in PGJ2-treated ZnDPP alone, at concentrations up to 30 M, to stimulated cul- cultures. tures did not affect viability nor change nitrite accumulation in the The Journal of Immunology 981

⌬12 medium (data not shown). In cultures treated with PGJ2, ZnDPP at 30 ␮M similarly did not affect viability (Fig. 5). In cultures treated with PG alone, nitrite accumulation was dose- dependently reduced, as seen previously (Fig. 1), with a 79% re- ␮ ⌬12 duction seen at 3 g/ml of PGJ2. Inclusion of ZnDPP partially ⌬12 reversed these effects, the inhibitory effect of PGJ2 being ap- proximately halved (43% reduction in nitrite accumulation with 3 ␮ ⌬12 g/ml PGJ2, compared with stimulated controls in the presence of this inhibitor).

Discussion This study represents the first work showing that naturally occur- ring prostaglandin PPAR␥ agonists are able to inhibit iNOS ac- tivity, measured by a reduction in nitrite accumulation and iNOS protein expression in macrophages, an effect that correlates with activation of HO-1. iNOS, inducible HO, the stress proteins, and the inducible Downloaded from COX-2 are several key physiologic and pathophysiologic systems that have stimulated much interest in recent years (2, 19). In par- ticular, the interplay between these systems has been the subject of much debate, not only because of the possibilities of using them as therapeutic targets, but also the possible ramifications of interfer- ing with any one of them. The recent discovery of the possibility of peroxisomal control of inflammation (15), by modulating the http://www.jimmunol.org/ destruction of key mediators of the inflammatory response such as FIGURE 3. Western blot analysis of the dose-dependent effects of the leukotrienes and other prostanoids, has offered another possi- ⌬12 PGJ2 on stimulated RAW264.7 macrophages. RAW264.7 murine mac- bility to the anti-inflammatory armory currently under develop- rophages (controls) were stimulated with 100 U/ml IFN-␥ and 100 ng/ml ment. To date, the effects of many of the PPAR agonists on iNOS, LPS (stimulated controls) for 18 h in the absence or presence of varying a key enzyme system in inflammation and other pathologies, have ⌬12 concentrations of PGJ2. Expression of iNOS, HO-1, HO-2, HSP70, and not been reported. GRP78 were assessed at this time by Western blotting. Initial studies investigated the role of the PPARs on the produc-

tion of NO by stimulated RAW264.7 macrophages, which are used by guest on September 24, 2021 in many in vitro studies (20). This was conducted using a variety

FIGURE 4. Western blot analysis of the time-depen- 12 dent effects of D PGJ2 on stimulated RAW264.7 mac- rophages. RAW264.7 murine macrophages were stimu- lated with 100 U/ml IFN-␥ and 100 ng/ml LPS atOhin the presence or absence of 3 ␮ 12 g/ml D PGJ2. At various times poststimulation (h) in- dicated above, Western blot analysis of iNOS, HO-1, HO-2, HSP70, and GRP78 was performed. 982 PPAR AGONISTS AND INHIBITION OF iNOS

cumulation. The reason behind this effect is unclear. However,

PGE2, the precursor of PGA2, has been shown to both increase iNOS activity at low concentrations and inhibit them at high con-

centrations in a similar fashion (25). PGD2 and it’s metabolites of ␥ the PGJ2 series are selective activators of the PPAR receptor pathway (26) and were demonstrated to inhibit iNOS. The data presented in this study suggest, therefore, that in these macro- phages, it is PPAR␥ activation that is responsible for the effects described. This adds to the previously published in vivo data sug- gesting that PPAR␣ activation is also an important pathway for anti-inflammatory activity (15). Further studies into the mechanism behind this inhibition used the technique of Western blotting to examine the levels of protein ex- ⌬12 pressed by these macrophages in response to treatment with PGJ2, the most potent inhibitor of nitrite accumulation activity investigated. Previously published work has revealed a link between these PGs and the induction of the stress response in a variety of cell lines (5, 12). We have previously published the observation that control of HO-1 Downloaded from activity can also affect the outcome of inflammation (4). In this study, it was shown that induction of HO-1 could markedly suppress an acute inflammatory reaction. In addition, work within this department has shown that the iNOS and HO-1 pathways exhibit a degree of interdependency (27). ⌬12 Examination of HO-1 expression elicited by PGJ2 revealed a correlation between the degree of inhibition of iNOS and the ex- http://www.jimmunol.org/ tent of induction of HO-1. Other markers of the stress response, HSP70 and GRP78, were unaffected by this treatment. Although HSP70 has been shown to be induced by these PGs in human lines, ⌬12 FIGURE 5. The effect of combined ZnDPP and PGJ2 treatment on this feature has been reported absent in murine cell lines (28). cell viability and nitrite production in RAW264.7 murine macrophages. Similarly, levels of HO-2, which has been described as a consti- RAW264.7 murine macrophages were stimulated with 100 U/ml IFN-␥ tutively expressed protein (29), were unchanged. There was no and 100 ␮g/ml LPS (controls) for 18 h in the presence of varying concen- ⌬12 correlation between the inhibition of iNOS and the expression of trations of PGJ2. Cell viability was measured by MTT reduction assay.

Nitrite production measured as accumulated product was assessed using COX-2, although cyclopentenone PGs have been shown to induce by guest on September 24, 2021 the Griess reaction. This was conducted with and without the presence of this enzyme in murine liver cell lines (30). This induction could 30 ␮M ZnDPP. **p Ͻ 0.01, ***p Ͻ 0.001 as measured by ANOVA with not be demonstrated in the current study using murine macro- post hoc Bonferroni-corrected t test, n ϭ 6 in each group; representative phages. The possibility that induction of COX-2 may play a role in data from three experiments. the anti-inflammatory properties of these PGs and/or PPAR acti- vation remains to be investigated. One possible mechanism in which this may be important may involve the generation of these

of naturally occurring eicosanoids, LTB4,8(S)-HETE, PGA2, “anti-inflammatory prostaglandins” at later time points in inflam- ⌬12 ␣ PGD2, PGJ2, and PG J2, as well as the PPAR -selective com- mation. In mast cells, COX-2 has been shown to exhibit different pound Wy14,643 to screen for their effects on nitrite production, modes of arachidonic acid acquisition compared with COX-1 and

indicative of activity of the iNOS pathway. LTB4 and 8(S)-HETE, is responsible for late phase, sustained release of PGD2 (31). This which activate PPAR␣ (15) and are described as poor activators of is the precursor for the PGJ series, which are formed spontane- ␥ PPAR , showed no inhibitory activity, but at high concentrations ously from PGD2 in biologic fluids in the presence of albumin caused a stimulation of nitrite accumulation. In the case of LTB4, (32), and may represent a negative feedback loop for inflammation this has been demonstrated for human neutrophils using a lower control by eicosanoid breakdown products. In the carrageenan ␮ dose of 0.1 M (21). In RAW264.7 macrophages, LTB4 has been pleurisy, Western blotting reveals two distinct peaks in protein

reported to have no effect on iNOS activity, although a concen- expression. An early peak is associated with PGE2 production, tration of only 1 nM was investigated (22). The ability to inhibit while a late peak is also found that is not associated with PGE2 nitrite accumulation of some of the other compounds examined elaboration (D.W., unpublished observations). The role of this lat- was shown not to be related to their cytotoxic potential, although ter peak in this model is the subject of current study, but it is this phenomenom was evident at higher concentrations. possible that this peak may represent a new function for COX-2 Wy14,643, also described as PPAR␣ selective (23), only inhibited during resolution by producing prostanoids that may help to ter-

nitrite accumulation, unlike LTB4 and 8(S)-HETE. It is therefore minate inflammation. possible that the stimulatory effects of these latter two are not The time courses of these changes in the level of these proteins related to PPAR␣ activation. Inhibition with Wy14,643 was, how- were constructed to attempt to further correlate any changes seen. ever, effective only at much higher concentrations than the PGs In non-PG-treated cells, iNOS protein was evident at6hand tested. It is known, however, that Wy14,643 is a less efficient peaked at 18 h, with a fall at 24 h, in agreement with previously PPAR␣-binding ligand and is capable of also activating PPAR␥ at published data (20). Inducible HO protein levels did not rise until the concentrations used here to inhibit nitrite accumulation (24). In 24 h; this correlated with the fall in iNOS protein expression seen contrast, the cyclopentenone PGs were effective inhibitors. The at the same time point. In PG-treated cells, the expression of HO-1

cyclopentenone PGA2 inhibited nitrite accumulation at higher con- was increased and also temporally shifted such that expression was centrations, but at lower concentrations PGA2 increased nitrite ac- seen much earlier, at 6 to 9 h. In keeping with a possible link The Journal of Immunology 983 between the two systems, iNOS induction was retarded until the and Wy14,643 are PPAR ligands and may exert their actions via HO-1 levels were reduced at 24 h. This latter finding has potential activation of these nuclear receptors. Together with the data pre- importance for the development of a therapeutic approach, due to sented in this paper, this suggests that the PPAR pathway may the short period of suppression of iNOS seen in these cultures. provide a novel and viable point of therapeutic intervention into However, it is known that the half-life of these PGs in vitro is the control of a wide variety of inflammatory diseases and other extremely short, and it is possible that HO-1 expression was main- pathologies. In particular, the efficacy of those ligands that have tained only for as long as they were present, with later escape of selectivity for PPAR␥ suggests that this is the pathway that may be the iNOS response due to the greater stability of the stimulus for of import in controlling certain aspects of the inflammatory iNOS in the culture system used. Again, no correlation was seen process.4 between the inhibition of iNOS and the expression of HSP70, GRP78, HO-2, or COX-2. References Given the possible link demonstrated between the induction of HO-1 and the inhibition of iNOS, the effect of ZnDPP, a specific 1. Vane, J. R. 1971. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat. New Biol. 231:232. HO-1 inhibitor (33), was examined in the whole cell system used 2. Appleton, I., A. Tomlinson, and D. A. Willoughby. 1995. Induction of cyclo- initially. ZnDPP was shown to have no effect on cell viability nor oxygenase and nitric oxide synthase in inflammation. Adv. Pharmacol. 35:27. on iNOS activity in this system in the absence of treatment with 3. Polla, B. S., and A. Cossarizza. 1996. Stress proteins in inflammation. In Stress- Inducible Cellular Responses. U. Feige, R. I. Morimoto, I. Yahara and B. Polla, the PG. However, when ZnDPP was included with the PG, the eds. Birkhauser Verlag, Basel, p. 375. inhibitory effect of the PG was reduced to half that seen in the 4. Willis, D., A. R. Moore, R. Frederick, and D. A. Willoughby. 1997. Heme ox- Downloaded from presence of the PG alone, suggesting that at least part of this in- ygenase: a novel target for the modulation of the inflammatory response. Nat. Med. 2:87. hibitory activity was due to activation of HO-1. The remainder 5. Amici, C., L. Sistonen, M. G. Santoro, and R. I. Morimoto. 1992. Antiprolifera- may represent a more direct action of the PG on iNOS induction, tive prostaglandins activate heat shock transcription factor. Proc. Natl. Acad. Sci. although to date no peroxisome proliferator response element has USA 89:6227. 6. Kliewer, S. A., J. M. Lenhard, T. M. Willson, I. Patel, D. C. Morris, and been described on the iNOS promoter. J. M. Lehmann. 1995. A prostaglandin J2 metabolite binds peroxisome proliferator- The precise mechanism whereby the interaction between HO-1 activated receptor gamma and promotes adipocyte differentiation. Cell 83:813. and iNOS is effected is unknown and will be the subject of further 7. Lemberger, T., B. 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