Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1103561 on February 24, 2004

Published online before print February 24, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1103561v1
75/6/1156    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leiro, J.
Right arrow Articles by Orallo, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Leiro, J.
Right arrow Articles by Orallo, F.
(Journal of Leukocyte Biology. 2004;75:1156-1165.)
© 2004 by Society for Leukocyte Biology

Effects of cis-resveratrol on inflammatory murine macrophages: antioxidant activity and down-regulation of inflammatory genes

José Leiro*, Ezequiel Álvarez{dagger}, Juan A. Arranz*, Reyes Laguna{dagger}, Eugenio Uriarte{ddagger} and Francisco Orallo{dagger},1

Departamentos de
* Microbiología y Parasitología,
{dagger} Farmacología, y
{ddagger} Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, Spain

1Correspondence: Departamento de Farmacología, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario Sur, E-15782 Santiago de Compostela (La Coruña), Spain. E-mail: fforallo{at}usc.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study investigated for the first time the effects of the cis isomer of resveratrol (c-RESV) on the responses of inflammatory murine peritoneal macrophages, namely on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during the respiratory burst; on the biosynthesis of other mediators of inflammation such prostaglandins; and on the expression of inflammatory genes such as inducible nitric oxide synthase (NOS)-2 and inducible cyclooxygenase (COX)-2. Treatment with 1–100 µM c-RESV significantly inhibited intracellular and extracellular ROS production, and c-RESV at 10–100 µM significantly reduced RNS production. c-RESV at 1–100 µM was ineffective for scavenging superoxide radicals (O2•–), generated enzymatically by a hypoxanthine (HX)/xanthine oxidase (XO) system and/or for inhibiting XO activity. However, c-RESV at 10–100 µM decreased nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase activity in macrophage homogenates. c-RESV at 100 µM decreased NOS-2 and COX-2 mRNA levels in lipopolysaccharide (LPS) interferon gamma (IFN-{gamma})-treated macrophages. At 10–100 µM, c-RESV also significantly inhibited NOS-2 and COX-2 protein synthesis and decreased prostaglandin E2 (PGE2) production. These results indicate that c-RESV at micromolar concentrations significantly attenuates several components of the macrophage response to proinflammatory stimuli (notably, production of O2•– and of the proinflammatory mediators NO• and PGE2).

Key Words: NADH/NADPH oxidase • xanthine oxidase • nitric oxide • inducible nitric oxide synthase • cyclooxygenase-2 • prostaglandin E2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of large-scale epidemiological studies have suggested that a prolonged and moderate consumption of red wine by the Southern French and other Mediterranean populations, despite a high-fat diet, little exercise, and widespread smoking, is associated with a very low incidence of cardiovascular diseases, mainly coronary heart disease (the so-called French paradox) [1 ].

The cardioprotective effects of red wine appear to be independent of alcohol content, and over the last few years, numerous studies have been performed with the aim of identifying the components responsible. Although these compounds have yet to be conclusively identified, the principal candidates are a number of polyphenolic compounds such as stilbenes and flavonoids (for review, see, e.g., ref. [2 ]).

Resveratrol [3,4',5-trihydroxystilbene (RESV)] is a natural phytoalexin (phytoestrogen) synthesized in response to injury or fungal attack, found in grape skin, and notably, in red wine in its two isomers, trans and cis (ref. [3 ]; see Fig. 1 ). A wide range of RESV concentrations in the different wines analyzed (depending on a number of factors) has been reported. In some wines, the cis isomer is in higher concentrations than the trans isomer, whereas in other wines, the predominant form is the trans-resveratrol (t-RESV; see, e.g., refs. [4 , 5 ]). To date, the majority of studies of pharmacological effects have considered t-RESV, which has been proposed as one of the principal candidates involved in the cardioprotective effects of red wine (see, e.g., ref. [6 ]). This isomer of RESV displays in the in vitro, ex vivo, and/or in vivo experiments a number of pharmacological effects including modulatory lipoprotein metabolism, anti-inflammatory, platelet antiaggregatory, anticarcinogenic, and vasodilatory properties, as has been reviewed previously (see, e.g., refs. [3 , 7 ]). Some of these pharmacological effects seem to be implicated in the cardiovascular protection attributed to t-RESV and red wine [7 ]. In contrast, much less is known about the pharmacological activity of the cis isomer (c-RESV), possibly as a result of the fact that c-RESV (unlike the trans isomer) is not available commercially. A few studies have demonstrated only quantitative differences in the activity of the two forms; for example, the cis isomer induces a greater decrease in collagen-induced platelet aggregation than the trans isomer [8 ], and in the cyclooxygenase (COX)-1 assay, t-RESV appears to be more active than c-RESV [9 ].



View larger version (7K):
[in this window]
[in a new window]
  		Figure 1. Generation of c-RESV after the irradiation of t-RESV with UV light (h{upsilon}).

 
During phagocytosis or stimulation with any of a wide variety of agents, macrophages activate various cell processes including the respiratory burst, in which an increase in oxygen uptake results in the production of reactive oxygen species (ROS), critical for successful defense against invading organisms [10 ]. ROS production occurs through two main mechanisms.

One is activation of a multicomponent nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase, which is assembled at the plasma membrane and cytosolic protein components and catalyzes the vectorial synthesis of superoxide radicals (O2•–) from oxygen and cellular NADPH (the preferred substrate in phagocytic cells), supplied by the hexose monophosphate shunt (see, e.g., ref. [11 ]).

The second is stimulation of the enzyme xanthine oxidase (XO), which catalyzes the oxidative hydroxylation of purine substrates (e.g., xanthine or hypoxanthine) at the molybdenum center (the reductive half-reaction) with production of uric acid and the subsequent reduction of O2 at the flavin center with generation of O2•– or H2O2 (the oxidative half-reaction) [12 , 13 ].

Increased ROS production by activated macrophages is also involved in immune diseases, and ROS excess (oxidant/antioxidant imbalance) may play an important role in the (patho)physiology associated with the progression of various chronic inflammatory disorders, such as atherosclerosis, rheumatoid arthritis, osteoarthritis, lung diseases, and neurodegenerative diseases [14 15 16 17 18 ]. In addition, during the respiratory burst, macrophages also produce nitric oxide (NO•), a major radical nitrogen species (RNS), which reacts with superoxide to form peroxynitrite, a potent microbicidal agent [19 ]. NO• and prostaglandin E2 (PGE2), produced by inducible NO• synthase type 2 (NOS-2) from the amino acid L-arginine and inducible COX-2, respectively, are also important mediators in inflammation processes [20 ]. In this context, a possible role of macrophage activation in different steps of coronary heart disease has been shown by several studies (see, e.g., refs. [21 22 23 ]). Because of its effects on activated macrophages and on other cell types, t-RESV has been proposed in a number of previous studies as one of the components present in red wine that may confer specific protection against ischemic heart disease [7 , 24 ].

To date, however, there have been no studies of the effects of c-RESV on macrophage respiratory burst responses or on production of inflammatory mediators. In the present study, we investigated the in vitro effects of c-RESV on ROS and RNS generation during the respiratory burst of murine thyoglicollate-elicited macrophages. Specifically, we investigated effects of c-RESV on the activities of the O2•–-generating enzymes NADH/NADPH oxidase and XO; whether c-RESV is able to scavenge O2•–; and whether c-RESV modulates NOS-2/COX-2 gene or protein expression and PGE2 production in inflammatory peritoneal macrophages stimulated with lipopolysaccharide (LPS) and interferon-{gamma} (IFN-{gamma}).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
The animals used throughout this study [Wistar rats (age 8–10 weeks; Iffa-Credo, L’Arbresle, Lyon, France), purchased from Criffa (Barcelona, Spain), and BALB/c mouse obtained from Harlan OLAC Ltd. (Oxon, UK)] were housed, cared for, and acclimatized (before the experiments) as previously indicated [6 ].

Ethical approval
All experiments were performed in accordance with European regulations (Directive 86/609), the Spanish Real Decreto 223/1988 and Orden Ministerial October/13/1989 on animal protection (Directive 86/609), and/or the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the U.S. National Institutes of Health (Bethesda, MD; NIH Publication No. 85-23, revised 1996). In addition, all experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Santiago de Compostela (Spain) and were conducted humanely.

Isolation of murine peritoneal-exudate macrophages
For induction of inflammatory responses, animals were injected intraperitoneally (i.p.) with 1 ml (rats) or 0.5 ml (mouse) 3% (w/v in water) thioglycollate broth, and peritoneal exudate was extracted 5 days later. Rat and mouse inflammatory peritoneal macrophages were obtained from animals killed by cervical dislocation in a laminar flow chamber to ensure sterile conditions, as described previously by Leiro et al. [25 , 26 ]. More than 97% of the adherent cells showed nonspecific esterase activity, determined as per Strober [27 ], indicating that they were macrophages.

Assay of ROS generation
The extracellular and intracellular production of ROS during the respiratory burst response of rat thioglycollate-elicited macrophages (i.e., macrophages from rats that had received thioglycollate broth i.p. 5 days previously) was evaluated with OxyBURST® Green probes (Molecular Probes, Leiden, The Netherlands) using the OxyBURST Green dihydro-2',4,5,6,7,7'-hexafluorofluorescein/bovine serum albumin (BSA) reagent for quantification of extracellular release of ROS induced by phorbol 12-myristate 13-acetate (PMA) and the amine-reactive OxyBURST Green succinimidyl ester of 2,7-dichloro-dihydrofluorescein (H2DCF) diacetate reagent conjugated to Kluyveromyces lactis yeast cells for quantitation of intracellular production of ROS as described previously [25 , 26 ].

Generation of O2•– by use of the enzymatic hypoxanthine oxidase (HX)–XO system
The O2•– was generated enzymatically in an HX–XO system and quantified by spectrophotometric measurement of the product of the reduction of nitroblue tetrazolium (NBT; Sigma, Alcobendas, Spain), essentially following the procedure described previously [6 , 26 ].

Determination of NADH/NADPH oxidase activity
Rat peritoneal macrophages that had been prestimulated in vivo with thioglycollate were obtained and counted as described previously [25 , 26 ], washed once by centrifugation at 1000 g for 10 min at 4°C, and resuspended in 1 ml Hanks’ balanced salt solution (HBSS) to an approximate concentration of 30–40 x 106 cells/ml. These cells, maintained on ice, were immediately lysed by sonication at 60 W for 1 min (two pulses of 30 s) to give the macrophage extract. Lysis was confirmed by light microscopy.

O2•– production was measured in this extract by lucigenin–enhanced chemiluminescence (ECL) as described by Orallo et al. [6 ] and Leiro et al. [26 ], using 100 µM NADPH (tetrasodium salt, Sigma; freshly diluted in HBSS) as substrate for NADH/NADPH oxidase.

Determination of XO activity by use of the xanthine–XO system
The potential effects of c-RESV on XO activity were tested by measuring its effects on uric acid formation from xanthine following the general method indicated in Orallo et al. [6 ].

Determination of XO activity by lucigenin–ECL assay
This assay was the same as the NADH/NADPH oxidase assay (see above) except that the NADH/NADPH oxidase substrate NADPH was replaced with the XO substrate xanthine (100 µM). The chemiluminescence signal emitted in these assays was approximately half that emitted with NADPH as substrate.

Assay of nitrite production
NO• production was estimated on the basis of nitrite production in rat macrophage culture supernatants, measured by the Griess reaction, as described elsewhere [25 , 28 ].

Determination of PGE2 in culture supernatants
Aliquots (100 µl) of the rat macrophage suspension (106 peritoneal macrophages per ml, obtained after prestimulation with thioglycollate as indicated above) were incubated for 3 h with LPS [100 ng/ml in Dulbecco’s modified Eagle medium (DMEM); Sigma] plus IFN-{gamma} (10 units/ml in DMEM); c-RESV at various concentrations (1, 10, or 100 µM in DMEM) or indomethacin (a known, nonselective inhibitor of COX expression and COX enzymatic activity; ref. [29 ]) at 30 µM; or without c-RESV or indomethacin (controls) in 96-well microculture plates at 37°C under 5% CO2 in a humidified incubator. The plates were centrifuged at 400 g for 5 min, and PGE2 levels were determined in the supernatant by using a competitive binding immunoassay (R & D Systems, Minneapolis, MN) following the manufacturer’s instructions.

Determination of NOS-2 and COX-2 mRNA levels by reverse transcriptase-polymerase chain reaction (RT-PCR)
Aliquots (1 ml) of 107 inflammatory rat peritoneal macrophages prestimulated in vivo 5 days previously with thioglycollate were incubated for 2 h at 37°C in DMEM containing 10 units/ml IFN-{gamma}, 100 ng/ml LPS, and c-RESV (1, 10, or 100 µM) or without c-RESV (controls). Total RNA was isolated from macrophage samples using a monophasic solution of phenol and guanidine thiocyanate (Tripure isolation reagent, Roche, Basel, Switzerland) following the manufacturer’s instructions. The resulting RNA was dried and then dissolved in diethylpyrocarbonate-treated RNase-free water at 1 µg/ml (Sigma). cDNA synthesis was accomplished as described previously [26 ]. The resulting cDNA (100 ng) was amplified by the PCR using a specific murine NOS-2 forward/reverse primer pair, 5'-TGGAAGCCGTAACAAAGGAAA-3'/5'-ACCACTCGTACTTGGGATGCT-3' [selected from the complete sequence of NOS-2 mRNA; GenBank of the National Center for Biotechnology Information (NCBI), accession number U03699], and a specific COX-2 forward/reverse primer pair, 5'-TGATCGAAGACTACGTGCAAC-3'/5'-TCATCTCTCTGCTCTGGTCAA-3' (NCBI, accession number NM_011198). Amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with the forward/reverse primer pair, 5'-ACCGCATCTTCTTGTGCAGT-3'/5'-GCCAAAGTTGTCATGGATGA-3' (NCBI, accession number AF106860), was performed as a control of gene expression. Thermal cycling conditions were as follows: initial denaturing at 94°C for 5 min; then, 35 cycles at 94°C for 30 s, 55°C (NOS-2 and COX-2) or 53°C (GAPDH) for 45 s, and 72°C for 1 min; and finally, a 7-min extension phase at 72°C. In all experiments, we performed RT-PCR controls without RNA or without RT; in no case were amplification products obtained. PCR products (20 µl aliquots) were separated on a 2% (w/v) agarose gel in Tris-boric acid–EDTA buffer, stained with 0.5 µg/ml ethidium bromide, and photographed with a digital camera under Spectroline 312 variable-intensity UV transilluminator (Spectroline, New York, NY). Bands were quantified in tagged image file format (TIFF) using densitometry analysis software (ImageMaster Total Laboratory, ver. 2.00, Amersham Biosciences Europe GmbH, Barcelona, Spain).

Slot-blot assay
The levels of the enzymes COX-2 and NOS-2 were determined by slot-blot assay in soluble extracts. Cultures of inflammatory mouse peritoneal macrophages (107 cells/ml) stimulated in vitro for 6 h in DMEM containing 10 units/ml IFN-{gamma}, 100 ng/ml LPS, and c-RESV at various concentrations (1, 10, and 100 µM) or without drug (controls) were incubated at 37°C in a humidified 5% CO2 atmosphere. After this incubation period, cells were washed with Tris-buffered saline (TBS: 50 mM Tris, 0.15 M NaCl, pH 7.4) and resuspended in 0.4 ml TBS and 0.1 ml protease inhibitor cocktail (Sigma) containing 104 mM 4-(2-amino-ethyl)-benzenesulfonyl fluoride, 80 µM aprotinin, 2 mM leupeptin, 4 mM bestatin, 1.5 mM pepstatin A, and 1.4 mM E-64 [N-(trans-epoxysuccinyl)-L-leucine-4-guanidinobutylamine]. Thereafter, the mouse peritoneal macrophages (maintained in ice) were sonicated at 60 W for 15 s and centrifuged for 5 min at 13,000 g, and the protein concentration in the supernatant was measured by the Bradford method [30 ] using BSA as standard. Equal amounts of total protein (60 µg in 100 µl) were transferred to a hydrophobic polyvinylidene difluoride membrane (Hybond-P, Amersham Biosciences) using a slot-blot filtration manifold (Amersham Biosciences). Membranes were subsequently blocked with 5% (w/v) nonfat milk powder in TBS containing 0.2% (w/v) Tween 20 for 1 h, and COX-2 and NOS-2 were immunodetected by incubation for 1 h at room temperature with a rabbit polyclonal antiserum against murine COX-2 (Cayman Chemical Co., Ann Arbor, MI) and NOS-2 (Alpha Diagnostic International, San Antonio, TX) diluted 1:1000 in dilution buffer [TBS containing 0.05% (w/v) Tween 20 and 1% (w/v) nonfat dry milk]. After incubation with primary antibody, the membranes were washed five times with washing buffer [TBS containing 0.05% (v/v) Tween 20], incubated for 1 h at room temperature with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin, Amersham Biosciences) at 1:3000 in dilution buffer, and washed five times with washing buffer. Finally, antibody binding was visualized by ECL detection (Amersham Biosciences) following the instructions from the supplier, using autoradiography film (Hyperfilm ECL, Amersham Biosciences). Bands in films were then scanned, stored as TIFF images, and analyzed by means of a densitometry analysis software (ImageMaster Total Laboratory, ver. 2.00, Amersham Biosciences). To convert relative light units (RLU; pixel intensity) into ng/mg protein, ovine COX-2 (Cayman Chemical Co.) and mouse NOS-2 peptide (a 19-amino acid peptide sequence designated INOS22-P; control peptide; 1125–1144 amino acids, Alpha Diagnostic International) were used as standards.

Stimuli, drugs, and chemicals
K. lactis (strain NRRL-41140) yeast cells were cultured on yeast extract–peptone–dextrose medium (Sigma) at 30°C at a growth rate of 0.07/h, then washed with distilled water, and finally freeze-dried. Stock solution of LPS from Escherichia coli serotype 0111:B4 (Sigma) was made up at 10 mg/ml in phenol-red-free DMEM (Sigma) containing 0.584 g/l glutamine (Sigma) and stored at –20°C until use. Thioglycollate broth (Merck, Darmstadt, Germany) was prepared to a concentration of 3% (w/v) in phosphate-buffered saline (PBS), autoclaved at 121°C for 10 min, and stored at room temperature until use. Recombinant murine IFN-{gamma} was purchased from Genzyme (Cambridge, MA). Stock solution (100 mM) of PMA (Sigma) was dissolved in dimethyl sulfoxide (DMSO) and stored in the dark at –80°C until use.

For preparation of c-RESV (Fig. 1 ), a solution of 500 mg t-RESV (Sigma) in 300 ml methanol was irradiated ({lambda}=254 nm) for 8 h. The solvent was then evaporated under reduced pressure, and the residue was purified by high-pressure liquid chromatography in a reverse-phase column (Cyclobond I2000 RSP, Astec, Whippany, NJ) using 1:1 methanol–water as eluent to give an oil, c-RESV (390 mg), and a white solid, t-RESV (100 mg). Retention times (flux of 1 ml/min in column of 500x9.2 mm): c-RESV, 10.4 min; t-RESV, 27.2 min.

1H nuclear magnetic resonance (MeOD) characterization of the c-RESV fraction: {delta} = 6.11 (t, 1H, J=2.1 Hz, H-4), 6.21 (d, 2H, J=2.1 Hz, H-2 and H-6), 6.31 (d, 1H, J=12.2 Hz, H-7), 6.41 (d, 1H, J=12.2 Hz, H-8), 6.62 (d, 2H, J=8.5 Hz, H-3' and H-5'), and 7.09 (d, 2H, J=8.5 Hz, H-2' and H-6') ppm.

Infrared characterization of the c-RESV fraction: {upsilon} = 3330, 1592, 1511, 1438, 1239, 1152, 1002, 835 cm–1.

Stock solutions (100 mM) of c-RESV and indomethacin (Sigma) were prepared in DMSO and stored in the dark at –80°C until use. Stock solutions of the arginine analog N-monomethyl-L-arginine monoacetate (L-NMMA, Calbiochem, San Diego, CA) and L(+) ascorbic acid (Merck) were made up at 100 mM in DMEM and stored until use at –20°C in the dark.

HX, sodium azide, xanthine, superoxide dismutase (SOD; from bovine erythrocytes), diphenyleneiodonium chloride (DPI), allopurinol, XO (from buttermilk), sulfanilamide, and naphthylenediamine hydrochloride were all purchased from Sigma. Appropriate dilutions of the above drugs were prepared every day immediately before use in PBS (XO assays), in HBSS (NADPH oxidase assays), and/or in deionized water (for the other experiments) from the following concentrated stock solutions kept at –20°C: SOD (20 kunits/ml) in deionized water, HX and xanthine (10 mM) in a 0.1% w/v aqueous KOH solution; all other compounds (100 mM) in deionized water. Xanthine oxidase was dissolved daily before the experiments in PBS. Deionized water and the appropriate dilutions of the different vehicles used had no significant, pharmacological effects in any of the tests performed in the present study. In addition, unlike t-RESV, c-RESV is a stable compound and never isomerized to the trans isomer during storage or throughout the period in which the different experiments were performed.

The specific chemicals and materials used in the different tests were purchased from suppliers indicated in the corresponding references provided in Materials and Methods. All other chemicals, including the reagents used in the preparation of different buffers and physiological solutions, were of the best quality commercially available.

Data presentation and statistical analysis
In figures, data are expressed as means ± SEM. Statistical comparisons ({alpha}=0.05) were performed by one-way ANOVA followed by the Tukey-Kramer test for multiple comparisons.

The details of the expression of results are specifically described in the references provided in the different Materials and Method sections.

The inhibitory effects of c-RESV and some of the reference drugs are expressed (where appropriate) as IC50 (concentrations that produce a 50% inhibition), calculated by least-squares linear regression using a fitting program (OriginTM 5.0, Microcal Software, Inc., Northampton, MA) of log molar concentration of the tested compound on percent of maximal pharmacological response obtained with each concentration.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of c-RESV on ROS production by inflammatory rat macrophages
The effects of c-RESV on extra- and intracellular ROS production by thioglycollate-prestimulated rat peritoneal macrophages were investigated using OxyBURST Green probes, and the results are shown in Figure 2 . Extracellular and intracellular ROS production in control inflammatory rat macrophages were, respectively, 81 ± 5 and 35 ± 2 arbitrary fluorescence units per min. c-RESV at concentrations of 1 µM or higher significantly inhibited PMA-induced extracellular ROS release, with an IC50 of 99 ± 8 µM (n=5; Fig. 2A ). Likewise, c-RESV at concentrations of 1 µM or higher significantly inhibited K. lactis-induced intracellular ROS production, with an IC50 of 14 ± 1.3 µM (n=5; Fig. 2B ).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Effects of c-RESV (1–100 µM) on the extracellular production of ROS induced by PMA (100 ng/ml; A) and on the intracellular production of ROS induced by K. lactis cells/OxyBURST® Green H2DCF complex (B) in thioglycollate-elicited rat peritoneal macrophages. L(+) ascorbic acid (0.1 mM; A) or sodium azide (1 mM; B) was used as an inhibitory reference drug. Each column represents the mean value ± SEM (indicated by vertical bars) for five experiments. Asterisks indicate significant differences (*, P<0.05, and **, P<0.01) with respect to control.

 
O2•–-scavenging capacity of c-RESV
We next investigated the ability of c-RESV to scavenge O2•– generated by an enzymatic system (HX–XO; Fig. 3 ). c-RESV itself was unable to directly reduce NBT. Control activity was 0.04 ± 0.003 A560 units/min (n=5). This value was unaffected by c-RESV (1–100 µM), and consequently, it had no effect as a selective scavenger of O2•– (Fig. 3) . SOD (0.1–10 units/ml), however, was capable of removing O2•– generated by the enzymatic HX–XO system (Fig. 3) . The IC50 value was 0.99 ± 0.08 units/ml (n=5).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 3. O2•– scavenging activity of c-RESV (1–100 µM) and the reference scavenger SOD (0.1–10 units/ml) in assays in which O2•– was generated by the enzymatic HX/XO system. Values shown are means ± SEM (n=5). *, P < 0.01, with respect to control values. A560nm, Absorbance at 560 nm.

 
Effects of c-RESV on XO activity
The effects of c-RESV on XO activity were investigated by measuring its effects on the production of uric acid from xanthine (assays using commercial XO; Fig. 4A ) or on O2•– production from xanthine as quantified by lucigenin–ECL (assays using rat macrophage homogenates as a source of XO; Fig. 4B ). c-RESV (1–100 µM) did not inhibit XO activity in either assay. By contrast, the reference XO inhibitor allopurinol at 10 µM significantly inhibited XO activity in both assays (Fig. 4) .



View larger version (20K):
[in this window]
[in a new window]
  		Figure 4. Effects of c-RESV (1–100 µM) and the XO inhibitor allopurinol (10 µM) on production of uric acid by XO in the presence of 100 µM xanthine (A) or on O2•– production from xanthine as quantified by lucigenin–ECL (B). Assays (A) were performed in vitro with commercial XO; assays (B) with an inflammatory rat macrophage homogenate as a source of XO. Values shown are mean ± SEM (n=5). *, P < 0.01, with respect to control.

 
Effects of c-RESV on NADH/NADPH oxidase activity in inflammatory rat macrophage homogenates
c-RESV itself was unable to reduce lucigenin directly. NADH/NADPH oxidase activity in control inflammatory rat macrophage homogenates was 5483 ± 411 RLU (n=5). c-RESV (10–100 µM) and DPI (1–100 µM), a know inhibitor of NADH/NADPH oxidase, decreased NADH/NADPH oxidase activity in a concentration-dependent manner, with IC50 values of 34 ± 3 and 28 ± 2 µM, respectively (n=5; Fig. 5 ).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 5. Effects of c-RESV (1–100 µM) and DPI (1–100 µM) NADH/NADPH oxidase activity (using NADPH 100 µM as substrate) in thioglycollate-elicited rat peritoneal macrophage homogenates as quantified by lucigenin–ECL. Values shown are mean ± SEM (n=5). *, P < 0.01, with respect to control values.

 
Effects of c-RESV on RNS production by inflammatory rat macrophages stimulated with LPS and IFN-{gamma}
Rat peritoneal macrophages elicited in vivo with thioglycollate and stimulated in vitro for 48 h with LPS and IFN-{gamma} were incubated in the presence or absence of c-RESV (1–100 µM). L-NMMA at 250 µM was used as reference inhibitor of NO• production. Cultures incubated with 10 or 100 µM c-RESV showed delayed and significantly reduced nitrite production, with an IC50 of 39 ± 3 µM (n=5; Fig. 6 ).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 6. In vitro effects of c-RESV (1–100 µM) and the NOS-2 inhibitor L-NMMA (250 µM) on NO• levels in supernatants of 48 h cultures of inflammatory rat peritoneal macrophages stimulated with 100 ng/ml LPS and 10 units/ml IFN-{gamma}. Values shown are means ± SEM (n=5). *, P < 0.01, with respect to control.

 
Effects of c-RESV on PGE2 production by inflammatory rat macrophages stimulated with LPS and IFN-{gamma}
We next investigated whether c-RESV inhibits the induction by LPS and IFN-{gamma} of PGE2 production in rat peritoneal macrophages. The synthesis of this active product of the COX pathway was inhibited by c-RESV (10–100 µM) and by the nonselective inhibitor of COX expression and COX enzymatic activity indomethacin at 30 µM (Fig. 7 ).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 7. Effects of c-RESV (1–100 µM) and indomethacin (30 µM, a known, nonselective inhibitor of COX expression and COX enzymatic activity) on PGE2 production by inflammatory rat peritoneal macrophages stimulated with 100 ng/ml LPS and 10 units/ml IFN-{gamma} for 3 h. Values shown are means ± SEM (n=5). *, P < 0.05, and **, P < 0.01, with respect to untreated control cells.

 
Effects of c-RESV on NOS-2 and COX-2 mRNA levels
To determine whether the above effects on NO• and PGE2 production are related to inhibitory effects in NOS-2 and COX-2 mRNA synthesis, we used a semiquantitative RT-PCR assay applied to RNA extracted from inflammatory rat peritoneal macrophages stimulated with LPS and IFN-{gamma}. Treatment with c-RESV (100 µM) significantly reduced levels of NOS-2 and COX-2 mRNA in the macrophage extracts (Fig. 8 ).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 8. Effects of c-RESV (10–100 µM) on NOS-2 and COX-2 mRNA levels in inflammatory rat peritoneal macrophages, as determined by semiquantitative RT-PCR. Macrophages were stimulated in vitro with LPS (100 ng/ml) and IFN-{gamma} (10 units/ml). (A) Agarose analysis (2%) of RT-PCR products using primers for NOS-2 (519 bp), COX-2 (345 bp), or the housekeeping gene GADPH (544 bp). (B) Densitometric quantification of normalized RT-PCR products of NOS-2 and COX-2 [optical density (OD) of enzyme band/OD of corresponding band in analysis using primer for GADPH]. Values are means ± SEM (n=5). *, P < 0.01, with respect to control values. Mw, Molecular weight markers (100 bp ladder).

 
Effects of c-RESV on synthesis of NOS-2 and COX-2
To determine the effect of c-RESV on NOS-2 and COX-2 protein levels in inflammatory peritoneal macrophages, a slot-blot analysis was used. As the corresponding rat antibodies are not commercially available (as far as we know) in these experiments, we used mouse peritoneal macrophages in place of rat macrophages. Treatment of LPS/IFN-{gamma}-stimulated, inflammatory macrophages with c-RESV (10–100 µM) significantly reduced the levels of NOS-2 (Fig. 9A ) and COX-2 (Fig. 9B) .



View larger version (34K):
[in this window]
[in a new window]
  		Figure 9. Effects of c-RESV (1–100 µM) on NOS-2 and COX-2 protein expression in inflammatory mouse peritoneal macrophages stimulated with LPS (100 ng/ml) and IFN-{gamma} (10 units/ml). Levels of NOS-2 (A) and COX-2 (B) were determined after 6 h of incubation by slot-blot assay; the figure shows a representative blot. Values shown are means ± SEM (n=5), expressed as ng enzyme per mg protein in the macrophage extract. *P < 0.01 with respect to control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have investigated for the first time the effects of c-RESV on inflammatory murine macrophage function.

During respiratory burst, macrophages produce and release large amounts of ROS and RNS, which play a role in the destruction of infectious agents; however, current evidence strongly indicates that activated macrophages are also involved in different steps of coronary heart disease (see Introduction) as well as in many inflammatory processes.

K. lactis and PMA rapidly stimulate macrophages to produce ROS [28 , 31 , 32 ] through stimulation of NADH/NADPH oxidase and/or XO enzymatic activity (see Introduction). c-RESV concentration-dependently inhibited extracellular and intracellular ROS production by inflammatory peritoneal macrophages at concentrations as low as 1 µM (i.e., it exhibited a typical antioxidant activity). Within the same range of concentrations (1–10 µM), t-RESV also inhibits ROS production by inflammatory rat peritoneal macrophages [28 ].

The above results may be attributable to effects on enzyme activities (XO and/or NADH/NADPH oxidase) or to direct scavenging of O2•– by c-RESV. We therefore first evaluated the O2•–-scavenging capacity of c-RESV, using the enzymatic HX–XO system. O2•– produced in this system reduces NBT to produce the colored compound formazan, which can be quantified spectrophotometrically as a measure of O2•– level [6 ]; in this assay system, c-RESV, like t-RESV [6 ], did not scavenge O2•–.

In view of these negative results, we investigated the possible effects of c-RESV on XO and/or NADH/NADPH oxidase enzymatic activity.

Like t-RESV (see ref. [6 ]), c-RESV did not modify the production of uric acid from xanthine (assays using commercial XO). In addition, c-RESV had no effects on O2•– generation from xanthine, as specifically quantified in the present study by lucigenin–ECL (assays using rat macrophage homogenates as source of XO). These data demonstrate that c-RESV does not affect XO activity.

By contrast, c-RESV inhibited NADH/NADPH oxidase enzymatic activity, i.e., the specific chemiluminescence signal emitted by the reaction between lucigenin and O2•– generated from NADPH, in inflammated rat macrophage extracts. These results indicate that the decrease of this enzymatic activity may be responsible, at least in part, for the inhibitory effects of c-RESV on ROS production. Similar effects on NADH/NADPH oxidase have been described previously for t-RESV in rat aortic homogenates [6 ].

Stimulation of murine macrophages with bacterial wall components (LPS) and IFN-{gamma} induces them to express large amounts of NOS-2, which leads to a strong, endogenous NO• production [33 , 34 ]. This NO• production is abrogated by 10–100 µM c-RESV (present results). Over the same concentration range, t-RESV likewise inhibits the production of nitrogen intermediates by inflammatory rat macrophages stimulated with LPS and IFN-{gamma} [28 ].

Activation of murine macrophages with LPS and IFN-{gamma} induces COX-2 expression and markedly increases PGE2 production [35 ], which is a potent mediator of inflammation and cell proliferation [36 ].

In the present study, treatment of LPS/IFN-{gamma}-stimulated inflammatory peritoneal macrophages with c-RESV inhibited PGE2 production, although only at concentrations of 10 µM or more. Similar results have been reported previously for t-RESV in mouse macrophages [37 ] and murine 3T6 fibroblast [38 ], which may explain, at least in part, the inhibition of the 3T6 fibroblast growth produced by this RESV isomer [38 ].

To investigate whether the above inhibitory effect of c-RESV on RNS and PGE2 production may be related (at least in part) to an action on NOS-2/COX-2 gene or protein expression, we also studied the potential effects of c-RESV on NOS-2/COX-2 mRNA and protein levels. Our results show that c-RESV at 100 µM inhibits NOS-2 and COX-2 mRNA production (determined by semiquantitative RT-PCR) in inflammatory rat peritoneal macrophages, and c-RESV at 10–100 µM inhibits NOS-2 and COX-2 protein synthesis (determined by slot-blot assay) in inflammatory mouse peritoneal macrophages treated with LPS and IFN-{gamma}. This apparent difference in effective inhibitory concentrations may be a result of the different animal models used (for justification of the choice of mouse macrophages in place of rat macrophages for the determination of NOS-2/COX-2 protein synthesis, see Results). Similar inhibitory effects have been described previously for t-RESV on NOS-2 mRNA levels [39 , 40 ] and on COX-2 enzyme expression [37 ] in mouse macrophages.

Taking into account that several studies have proposed a positive modulatory role of PGE2 (see, e.g., ref. [41 ]) and ROS [37 , 42 ] on NOS-2 induction in murine macrophages, the inhibitory effects of c-RESV on NOS-2 mRNA and protein levels could be related, at least in part, to the ability of this RESV isomer for decreasing the production of ROS and PGE2. Furthermore, as previous studies have shown that NO• is necessary for maintaining prolonged COX-2 gene expression (see, e.g., ref. [35 ]), the action of c-RESV on RNS production observed in the present study could be responsible, at least in part, for the effects of this natural compound on COX-2 mRNA and enzyme levels.

Moreover, in the same way as t-RESV in mice [39 , 43 , 44 ] or in humans (see, e.g., ref. [45 ]), the action of the c-RESV on NOS/COX-2 gene and protein expression described above could be mediated by an inhibition of the nuclear factor-{kappa}B (NF-{kappa}B) activation, mainly as a result of the inhibition of the degradation of inhibitor of {kappa}B{alpha} (I{kappa}B{alpha}). The precise effects of c-RESV on the NF-{kappa}B signaling pathway are currently being evaluated in our laboratory, basically using a DNA hybridization array containing 96 NF-{kappa}B-related genes. The preliminary results obtained in these experiments (data not shown) indicate that c-RESV produces an increase of the I{kappa}B gene expression and a down-regulation of two genes of the NF-{kappa}B family, NF-{kappa}B1 and NF-{kappa}B2.

In summary, our data indicate that c-RESV at micromolar concentrations significantly attenuates several components of the macrophage response to proinflammatory stimuli. Specifically, c-RESV blocks O2•– production by inflammatory macrophages (by inhibition of NADPH/NADPH oxidase activity) and inhibits the production of the proinflammatory mediators NO• and PGE2 (at least in part by inhibition of NOS-2 and COX-2 gene and protein expression). These inhibitory effects on the inflammatory response are qualitatively similar to those previously observed for t-RESV. Therefore, the different spatial conformation of c-RESV (vs. that of the trans isomer, see Fig. 1 ) does not seem to modify markedly its interaction with the potential cellular targets.

As noted in Introduction, because of its effects on macrophage function and on other cell types, t-RESV has been implicated in the protection induced by the prolonged consumption of moderate amounts of wine, especially of red wine, against the incidence of cardiovascular diseases (mainly coronary heart disease). In this context, it has been described previously that the t-RESV concentrations reached in plasma and tissues after the oral administration of this natural compound to rats and humans seem to approach the in vitro active concentrations; an average drinker of wine can, particularly in the long term, absorb a sufficient amount of RESV to explain the beneficial effect of red wine on health (see, e.g., refs. [46 , 47 ]).

In addition, it is interesting to point out (see also Introduction) that an increased macrophage activation (e.g., through, among others, overproduction of ROS and RNS, abnormally high NADH/NADPH oxidase, NOS-2, and/or COX-2 activities) has been observed in different steps of coronary heart disease and in a number of inflammatory processes (including atherosclerosis). Bearing in mind the above considerations and assuming that c-RESV shows a similar behavior in humans, our results suggest that the beneficial cardioprotective effects of consumption of foods and beverages containing RESV (notably red wine) may thus be a result of the combined effects of the two isomers.

Finally, taking into account the pharmacological properties described for c-RESV in the present study, it can also be concluded that this natural polyphenol may have value as a structural model for the design and subsequent development of new drugs useful for improving the pharmacological treatment of inflammatory diseases involving hyper-responsiveness of phagocytic cells.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT; Spain; SAF2000-0137 and SAF2002-02645), the Consellería de Educación e Ordenación Universitaria, Xunta de Galicia (Spain; PGIDIT02BTF20301PR and PGIDIT02PXIC20305PN), and Almirall Prodesfarma Laboratories (Spain). In addition, this study was awarded with the 2003 prize of the Spanish Pharmacological Society and Almirall Prodesfarma Laboratories.

Received November 13, 2003; revised January 8, 2004; accepted January 15, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Renaud, S., de Lorgeril, M. (1992) Wine, alcohol, platelets, and the French paradox for coronary heart disease Lancet 339,1523-1526[CrossRef][Medline]
  2. Duffy, S. J., Vita, J. A. (2003) Effects of phenolics on vascular endothelial function Curr. Opin. Lipidol. 14,21-27[CrossRef][Medline]
  3. Soleas, G. J., Diamandis, E. P., Goldberg, D. M. (2001) The world of resveratrol Adv. Exp. Med. Biol. 492,159-182[Medline]
  4. Soleas, G. J., Diamandis, E. P., Goldberg, D. M. (1997) Resveratrol: a molecule whose time has come? And gone? Clin. Biochem. 30,91-113[CrossRef][Medline]
  5. Burns, J., Gardner, P.T., O’Neil, J., Craeford, S., Morecroft, I., Mcphail, D. B., Lister, C., Matthews, D., Mclean, M. R., Lean, M. E. J., Duthie, G. G., Crozier, A. (2000) Relationship among antioxidant activity, vasodilatation capacity, and phenolic content of red wines J. Agric. Food Chem. 48,220-230[CrossRef][Medline]
  6. Orallo, F., Álvarez, E., Camiña, M., Leiro, J. M., Gómez, E., Fernández, P. (2002) The possible implication of trans-resveratrol in the cardioprotective effects of long-term moderate wine consumption Mol. Pharmacol. 61,294-302[Abstract/Free Full Text]
  7. Wu, J. M., Wang, Z. R., Hsieh, T. C., Bruder, J. L., Zou, J. G., Huang, Y. Z. (2001) Mechanism of cardioprotection by resveratrol, a phenolic antioxidant present in red wine Int. J. Mol. Med. 8,3-17[Medline]
  8. Bertelli, A. A., Giovannini, L., Bernini, W., Migliori, M., Fregoni, M., Bavaresco, L., Bertelli, A. (1996) Antiplatelet activity of cis-resveratrol Drugs Exp. Clin. Res. 22,61-63[Medline]
  9. Waffo-Teguo, P., Hawthorne, M. E., Cuendet, M., Merillon, J. M., Kinghorn, A. D., Pezzuto, J. M., Mehta, R. G. (2001) Potential cancer-chemopreventive activities of wine stilbenoids and flavans extracted from grape (Vitis vinifera) cell cultures Nutr. Cancer 40,173-179[CrossRef][Medline]
  10. Shepherd, V. L. (1986) The role of the respiratory burst of phagocytes in host defense Semin. Respir. Infect. 1,99-106[Medline]
  11. Forman, H. J., Torres, M. (2002) Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling Am. J. Respir. Crit. Care Med. 166,S4-S8
  12. Rinaldo, J. E., Clark, M., Parinello, J., Shepherd, V. L. (1994) Nitric oxide inactivates xanthine dehydrogenase and xanthine oxidase in interferon-{gamma}-stimulated macrophages Am. J. Respir. Cell Mol. Biol. 11,625-630[Abstract]
  13. Cos, P., Ying, L., Calomme, M., Hu, J. P., Cimanga, K., Van Poel, B., Pieters, L., Vlietinck, A. J., Berghe, D. V. (1998) Structure-activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers J. Nat. Prod. 61,71-76[CrossRef][Medline]
  14. Dusting, G. J., Fennessy, P., Yin, Z. L., Gurevich, V. (1998) Nitric oxide in atherosclerosis: vascular protector or villain? Clin. Exp. Pharmacol. Physiol. Suppl. 25,S34-S41[Medline]
  15. Floyd, R. A. (1999) Antioxidants, oxidative stress, and degenerative neurological disorders Proc. Soc. Exp. Biol. Med. 222,236-245[Abstract/Free Full Text]
  16. Agostini, M., Di Marco, B., Nocentini, G., Delfino, D. V. (2002) Oxidative stress and apoptosis in immune diseases Int. J. Immunopathol. Pharmacol. 15,157-164[Medline]
  17. Chernajovsky, Y., Winyard, P. G., Kabouridis, P. S. (2002) Advances in understanding the genetic basis of rheumatoid arthritis and osteoarthritis: implications for therapy Am. J. Pharmacogenomics 2,223-234[CrossRef][Medline]
  18. Comhair, S. A., Erzurum, S. C. (2002) Antioxidant responses to oxidant-mediated lung diseases Am. J. Physiol. Lung Cell. Mol. Physiol. 283,L246-L255[Abstract/Free Full Text]
  19. Knight, J. A. (2000) Review: free radicals, antioxidants, and the immune system Ann. Clin. Lab. Sci. 30,145-158[Abstract]
  20. Weinberg, J. B. (2000) Nitric oxide synthase 2 and cyclooxygenase 2 interactions in inflammation Immunol. Res. 22,319-341[CrossRef][Medline]
  21. Mazzone, A., De Servi, S., Ricevuti, G., Mazzucchelli, I., Fossati, G., Pasotti, D., Bramucci, E., Angoli, L., Marsico, F., Specchia, G., Notario, A. (1993) Increased expression of neutrophil and monocyte adhesion molecules in unstable coronary artery disease Circulation 88,358-363[Abstract/Free Full Text]
  22. Auer, J., Berent, R., Labetanig, E., Eber, B. (2001) Serum neopterin and activity of coronary artery disease Heart Dis. 3,297-301[CrossRef][Medline]
  23. Ikeda, U., Matsui, K., Murakami, Y., Shimada, K. (2002) Monocyte chemoattractant protein-1 and coronary artery disease Clin. Cardiol. 25,143-147[Medline]
  24. Wallerath, T., Deckert, G., Ternes, T., Anderson, H., Li, H., Witte, K., Forstermann, U. (2002) Resveratrol, a polyphenolic phytoalexin present in red wine, enhances expression and activity of endothelial nitric oxide synthase Circulation 106,1652-1658[Abstract/Free Full Text]
  25. Leiro, J., Iglesias, R., Paramá, A., Sanmartín, M. L., Ubeira, F. M. (2001) Respiratory burst responses of rat macrophages to microsporidian spores Exp. Parasitol. 98,1-9[CrossRef][Medline]
  26. Leiro, J. M., Álvarez, E., Arranz, J. A., Siso, I. G., Orallo, F. (2003) In vitro effects of mangiferin on superoxide concentrations and expression of the inducible nitric oxide synthase, tumour necrosis factor-{alpha} and transforming growth factor-ß genes Biochem. Pharmacol. 65,1361-1371[CrossRef][Medline]
  27. Strober, W. (1997) Wright-Giemsa and nonspecific esterase staining of cells Coligan, J. E. Kruissbeek, A. M. Margulies, D. H. Shevach, E. M. Strober, W. eds. Current Protocols in Immunology 3,A.3C.1-A.3C.2 John Wiley & Sons New York, NY.
  28. Leiro, J., Álvarez, E., García, D., Orallo, F. (2002) Resveratrol modulates rat macrophage functions Int. Immunopharmacol. 2,767-774[Medline]
  29. Kato, M., Nishida, S., Kitasato, H., Sakata, N., Kawai, S. (2001) Cyclooxygenase-1 and cyclooxygenase-2 selectivity of non-steroidal anti-inflammatory drugs: investigation using human peripheral monocytes J. Pharm. Pharmacol. 53,1679-1685[CrossRef][Medline]
  30. Bradford, M. M. (1976) A rapid and sensitive method for the quantification of microgram quantitites of protein using the principle of protein-dye binding Anal. Biochem. 72,248-254[CrossRef][Medline]
  31. Álvarez, E., Leiro, J., Orallo, F. (2002) Effect of (–)-epigallocatechin-3-gallate on respiratory burst of rat macrophages Int. Immunopharmacol. 2,849-855[CrossRef][Medline]
  32. García, D., Delgado, R., Ubeira, F. M., Leiro, J. (2002) Modulation of rat macrophage function by the Mangifera indica L. extracts Vimang and mangiferin Int. Immunopharmacol. 2,797-806[CrossRef][Medline]
  33. Virgili, F., Kobuchi, H., Packer, L. (1998) Procyanidins extracted from Pinus maritima (pycnogenol): scavengers of free radical species and modulators of nitrogen monoxide metabolism in activated murine RAW 264.7 macrophages Free Radic. Biol. Med. 24,1120-1129[CrossRef][Medline]
  34. Ferret, P. J., Soum, E., Negre, O., Fradelizi, D. (2002) Auto-protective redox buffering systems in stimulated macrophages BMC Immunol. 3,3[CrossRef][Medline]
  35. Perkins, D. J., Kniss, D. A. (1999) Blockade of nitric oxide formation down-regulates cyclooxygenase-2 and decreases PGE2 biosynthesis in macrophages J. Leukoc. Biol. 65,792-799[Abstract]
  36. Bagga, D., Wang, L., Farias-Eisner, R., Glaspy, J. A., Reddy, S. T. (2003) Differential effects of prostaglandin derived from {omega}-6 and {omega}-3 polyunsaturated fatty acids on COX-2 expression and IL-6 secretion Proc. Natl. Acad. Sci. USA 100,1751-1756[Abstract/Free Full Text]
  37. Martínez, J., Moreno, J. J. (2000) Effect of resveratrol, a natural polyphenolic compound, on reactive oxygen species and prostaglandin production Biochem. Pharmacol. 59,865-870[CrossRef][Medline]
  38. Moreno, J. J. (2000) Resveratrol modulates arachidonic acid release, prostaglandin synthesis, and 3T6 fibroblast growth J. Pharmacol. Exp. Ther. 294,333-338[Abstract/Free Full Text]
  39. Tsai, S. H., Lin-Shiau, S. Y., Lin, J. K. (1999) Suppression of nitric oxide synthase and the down-regulation of the activation of NF{kappa}B in macrophages by resveratrol Br. J. Pharmacol. 126,673-680[CrossRef][Medline]
  40. Chan, M. M., Mattiacci, J. A., Hwang, H. S., Shah, A., Fong, D. (2000) Synergy between ethanol and grape polyphenols, quercetin, and resveratrol, in the inhibition of the inducible nitric oxide synthase pathway Biochem. Pharmacol. 60,1539-1548[CrossRef][Medline]
  41. Chen, B. C., Lin, W. W. (2000) Pyrimidinoceptor potentiation of macrophage PGE2 release involved in the induction of nitric oxide synthase Br. J. Pharmacol. 130,777-786[CrossRef][Medline]
  42. Martínez, J., Sánchez, T., Moreno, J. J. (2000) Regulation of prostaglandin E2 production by the superoxide radical and nitric oxide in mouse peritoneal macrophages Free Radic. Res. 32,303-311[CrossRef][Medline]
  43. Wadsworth, T. L., Koop, D. R. (1999) Effects of the wine polyphenolics quercetin and resveratrol on pro-inflammatory cytokine expression in RAW 264.7 macrophages Biochem. Pharmacol. 57,941-949[CrossRef][Medline]
  44. Murakami, A., Matsumoto, K., Koshimizu, K., Ohigashi, H. (2003) Effects of selected food factors with chemopreventive properties on combined lipopolysaccharide- and interferon-{gamma}-induced I{kappa}B degradation in RAW264.7 macrophages Cancer Lett. 195,17-25[CrossRef][Medline]
  45. Adhami, V. M., Afaq, F., Ahmad, N. (2003) Suppression of ultraviolet B exposure-mediated activation of NF-{kappa}B in normal human keratinocytes by resveratrol Neoplasia 5,74-82[Medline]
  46. Bertelli, A., Bertelli, A. A. E., Gozzini, A., Giovannini, L. (1998) Plasma and tissue resveratrol concentrations and pharmacological activity Drugs Exp. Clin. Res. 24,133-138[Medline]
  47. Soleas, G. J., Yan, J., Goldberg, D. M. (2001) Ultrasensitive assay for three polyphenols (catechin, quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection J. Chromatogr. B Biomed. Sci. Appl. 757,161-172[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
C.-C. Lu, H.-C. Lai, S.-C. Hsieh, and J.-K. Chen
Resveratrol ameliorates Serratia marcescens-induced acute pneumonia in rats
J. Leukoc. Biol., April 1, 2008; 83(4): 1028 - 1037.
[Abstract] [Full Text] [PDF]

Home page
 Therapeutic Advances in Respiratory DiseaseHome page
A. Sharafkhaneh, S. Velamuri, V. Badmaev, C. Lan, and N. Hanania
Review: The potential role of natural agents in treatment of airway inflammation
Therapeutic Advances in Respiratory Disease, December 1, 2007; 1(2): 105 - 120.
[Abstract] [PDF]

Home page
 Int ImmunolHome page
S. J. Prasanna, B. Saha, and D. Nandi
Involvement of oxidative and nitrosative stress in modulation of gene expression and functional responses by IFN{gamma}
Int. Immunol., July 2, 2007; (2007) dxm058v1.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
S.-E. Chow, Y.-C. Hshu, J.-S. Wang, and J.-K. Chen
Resveratrol attenuates oxLDL-stimulated NADPH oxidase activity and protects endothelial cells from oxidative functional damages
J Appl Physiol, April 1, 2007; 102(4): 1520 - 1527.
[Abstract] [Full Text] [PDF]

Home page
 Plant Cell PhysiolHome page
C. K. Y. Yu, C. N. W. Lam, K. Springob, J. Schmidt, I. K. Chu, and C. Lo
Constitutive Accumulation of cis-piceid in Transgenic Arabidopsis Overexpressing a Sorghum Stilbene Synthase Gene
Plant Cell Physiol., July 1, 2006; 47(7): 1017 - 1021.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1103561v1
75/6/1156    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Leiro, J.
Right arrow Articles by Orallo, F.