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Resveratrol modulates innate and inflammatory responses

in fish leucocytes

R. Castro a, J. Lamas a, P. Morais b, M.L. Sanmartın b,F. Orallo c, J. Leiro b,*

a Departamento de Biologıa Celular y Ecologıa, Facultad de Biologıa, Universidad de Santiago de Compostela,

15782 Santiago de Compostela, Spainb Departamento de Microbiologıa y Parasitologıa, Laboratorio de Parasitologıa, Instituto de Investigacion y Analisis Alimentarios,

Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spainc Departamento de Farmacologıa, Facultad de Farmacia, Universidad de Santiago de Compostela,

15782 Santiago de Compostela, Spain

Received 24 February 2008; received in revised form 23 May 2008; accepted 5 June 2008

Abstract

Resveratrol (RESV; trans-3,5,4’-trihydroxystilbene), a phytoalexin that is produced by some plants, among other effects has

well-known antioxidant, anti-inflammatory and immunomodulatory activities in mammals. In the present study, the effects of

RESVon several functions of turbot, Psetta maxima (L.), kidney leucocytes (KLs) related to the innate and inflammatory responses

were investigated. RESV exerted a dose-dependent inhibitory effect on the migratory response and on the production of reactive

oxygen species in KL, after stimulation of the respiratory burst activity with phorbol myristate acetate (PMA). RESV also

significantly inhibited the generation of the pro-inflammatory mediator prostaglandin E2 (PGE2) in the supernatant of KL cultures

stimulated with acidic sulphated polysaccharides (ASPs) from the seaweed Ulva rigida. The effects of the polyphenol on enzymatic

activity and on myeloperoxidase (MPO) gene expression in neutrophils were also tested. It was found that RESV strongly inhibited

intracellular and extracellular MPO activity, behaving as a noncompetitive and reversible inhibitor, and also induced a decrease in

MPO mRNA levels in turbot neutrophils. These findings indicate that RESV exerts important modulatory effects on inflammatory

responses in fish, and considering the importance of innate immunity in these vertebrates and the similarities with mammals, it may

be possible to use fish for analysis of the effects of different substances on inflammatory responses.

# 2008 Elsevier B.V. All rights reserved.

Keywords: Resveratrol; Turbot leucocytes; Innate immune response; Myeloperoxidase; Inflammation; Respiratory burst

1. Introduction

Polyphenols are a group of phytochemicals that are

increasingly considered as being responsible for the

health benefits provided by fruit and vegetables (Fraga,

2007). Resveratrol (RESV; trans-3,5,4’-trihydroxystil-

bene), a natural polyphenol, was first isolated in 1940 as

a constituent of the roots of white hellebore (Veratrum

grandiflorum O. Loes), but since then it has been found

in various plants, including grapes, berries and peanuts

(Khanna et al., 2007). RESV has a variety of effects on

mammals including protection against ischaemia-

reperfusion injury, as well as antitumour and chemo-

preventative action against malignant tumours (Chen

www.elsevier.com/locate/vetimm

Available online at www.sciencedirect.com

Veterinary Immunology and Immunopathology 126 (2008) 9–19

* Corresponding author at: Laboratorio de Parasitologıa, Instituto de

Investigacion y Analisis Alimentarios, Universidad de Santiago de

Compostela, C/ Constantino Candeira s/n, 15782 Santiago de Com-

postela, Spain. Tel.: +34 981563100; fax: +34 981547171.

E-mail address: mpleiro@usc.es (J. Leiro).

0165-2427/$ – see front matter # 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetimm.2008.06.001

Author's personal copy

and Tseng, 2007); and it has recently been demonstrated

that RESV extends lifespan in fish (Valenzano and

Cellerino, 2006).

Fish represent the earliest class of vertebrates that

possess elements of both innate and acquired immunity,

and are considered of great interest for study of the

similarities and differences between their defence

mechanisms and those of higher vertebrates (Plouffe

et al., 2005). In fish, the innate immune response has

been considered an essential component in combating

disease due to the constraints placed on the adaptive

immune response in terms of the limited antibody

repertoires, affinity maturation and memory and

relatively slow lymphocyte proliferation (Pedrera

et al., 1992; Collazos et al., 1995; Magnadottir, 2006;

Whyte, 2007). Although inflammation constitutes a

primary response of an organism’s defence mechanism,

inflammation may also underlie various chronic

diseases (Kundu et al., 2004). Both acute and chronic

inflammatory responses occur through cellular reac-

tions mediated by chemical factors such as cytokines,

chemokines, prostaglandins (PGs), nitric oxide (NO)

and other reactive oxygen species (ROS), leukotrienes,

etc., which are produced in response to pro-inflamma-

tory stimuli (Kumar et al., 2004). In animals, including

fish, the inflammatory response to various infectious

agents involves strong stimulation of the polymorpho-

nuclear neutrophils that release ROS and myeloperox-

idase (MPO), and inhibition of the oxidising and MPO

activities of neutrophils offers therapeutic perspectives

in animal pathologies with excessive inflammatory

reactions (Iglesias et al., 2001; Kohnen et al., 2007;

Stakauskas et al., 2007). RESV is an important

inhibitory agent of inflammatory processes, which in

mammals are regulated through the NF-kappa-B

pathway (Leiro et al., 2003, 2004a,b, 2005; Nam,

2006). Because RESV has an inhibitory effect on the

production of ROS by mammalian neutrophils (Caval-

laro et al., 2003; Kohnen et al., 2007), it is possible that

this polyphenol may decrease the inflammatory

response, and that the prolonged lifespan observed in

fish may be at least partly associated with a decrease in

the amount of ROS produced.

The present study was carried out to determine

whether RESV affects the inflammatory response in

turbot, Psetta maxima (L.), by analysis of migration,

production of prostaglandins, respiratory burst activity

and MPO activity and gene expression in fish kidney

leucocytes (KLs). We used turbot because this is one of

the most important fish species in European aqua-

culture, because we have previously studied all these

activities in kidney leucocytes response to different

stimuli and finally because we have characterized turbot

MPO and sequenced a fragment of the gene (Castro

et al., 2008), thereby facilitating the experimental

analyses involved.

2. Materials and methods

2.1. Fish

Male and female turbot (50–100 g; about 3 months

old) were obtained from a local fish farm in Galicia

(northwestern Spain). Prior to experiments, the fish

were acclimatized for at least 15 days in 200 l tanks with

a constant flow of water (14 8C, pH 6.5 � 0.5) and

aeration. The fish were fed daily (between 10:00 and

11:00 a.m.) with a standard, commercially available

semi-dried pelleted food.

2.2. Ethical approval

All experiments were carried out in accordance with

European regulations on animal protection (Directive

86/609), agreed under the Declaration of Helsinki. All

experimental protocols were approved by the Institu-

tional Animal Care and Use Committee of the

University of Santiago de Compostela (Spain).

2.3. Stimuli, drugs, and chemicals

Strain I1 of the ciliate Philasterides dicentrarchi

(Iglesias et al., 2001) was maintained under the culture

conditions described (Iglesias et al., 2003). Turbot was

experimentally infected by intraperitoneal inoculation

of 5 � 104 ciliates and the fish developed fluid in the

abdominal cavity 4 days post-inoculation (Parama et al.,

2004a,b). Ascitic fluid was obtained by abdomenocent-

esis and separation of ciliates by centrifugation at

600 � g for 5 min; the fluid was aliquoted and stored at

�80 8C until use.

Parasite lysate was obtained as described (Parama

et al., 2004a,b). Briefly, the ciliates were washed three

times by centrifugation at 600 � g for 5 min in 0.25 M

sucrose at 4 8C. The cells were then disrupted

ultrasonically at 4 8C in a protease inhibitor cocktail

(Sigma–Aldrich). The homogenate was centrifuged at

15,000 � g for 15 min at 4 8C and aliquots of the

resultant supernatant fraction were stored at �80 8C.

Proteases from P. dicentrarchi trophozoites were

purified on a bacitracin-sepharose affinity column, as

previously described (Parama et al., 2007). Aliquots of

the 2.5 ml fractions collected were assayed for

proteolytic activity, as previously described (Parama

R. Castro et al. / Veterinary Immunology and Immunopathology 126 (2008) 9–1910

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et al., 2004a,b) and those fractions showing enzyme

activity were pooled and concentrated by ultrafiltration

with Amicon1 Ultra K centrifugal filter devices

(Millipore, USA), dissolved in 0.15 M PBS and stored

at �80 8C until use. The concentration of protein in the

ascitic fluid and in ciliate lysates was determined by the

Bradford method, with a Bio-Rad Protein Assay kit

(Bio-Rad Laboratories, Germany), and with bovine

serum albumin (BSA; Sigma–Aldrich, Spain) as

standard.

Acidic sulphated polysaccharides (ASPs) were

obtained from the seaweed Ulva rigida C. Agardh

and analysed for monosaccharide composition and

structure, as previously described (Castro et al., 2004,

2006; Leiro et al., 2007).

A stock solution (100 mM) of phorbol myristate

acetate (PMA; Sigma–Aldrich), trans-resveratrol

(RESV; Sigma–Aldrich) and indomethacin (INDO;

Sigma–Aldrich) was prepared in dimethyl sulphoxide

(DMSO) and stored in the dark at�80 8C until use. L(+)

Ascorbic acid (Sigma–Aldrich) was prepared in PBS

and stored at �20 8C until use.

2.4. Isolation of turbot kidney leucocytes and

preparation of cell lysates

The kidney is main haematopoietic organ in turbot.

Isolation of kidney leucocytes was carried out as

described by Castro et al. (2004, 2006). Briefly, cells

were suspended in L-15 medium with 0.1% FCS at a

concentration of 2 � 107 viable cells per millilitre

(determined by the trypan blue exclusion test). The

medium was carefully removed before starting the

experiments to obtain a mixed culture of leucocytes,

containing about 50% of peroxidase-positive cells

(neutrophils and neutrophil precursors) and 12% non-

esterase positive cells (monocytes–macrophages).

Before carrying out the experiments, the mean number

of cells per well was estimated by counting the number

of nuclei present after lysing the cells in several wells

with 100 ml lysis buffer containing 0.1 M citric acid, 1%

Tween 20 and 0.05% crystal violet (Castro et al., 2004).

Kidney leucocyte pellets containing 109 cells were

suspended in 8 ml ice-cold disruption buffer [50 mM

Tris–HCl, pH 7.5, containing 0.5 M NaCl, 1 mM

EDTA, and proteinase inhibitors (100 mg/ml phenyl-

methylsulphonyl fluoride (PMSF), 2 mg/ml aprotinin,

2 mg/ml leupeptin)], and were lysed by sonication

(eight bursts each of 1 min duration) on ice. The lysate

was then centrifuged at 12,000 � g for 30 min, and the

supernatant was stored at �80 8C until use. The protein

concentration of the supernatant was determined with

the Bio-Rad Protein Assay kit (Bio-Rad Laboratories,

Germany), as indicated above.

2.5. Purification of myeloperoxidase (MPO)

Turbot neutrophil MPO was purified from KL lysates

on a chromatography column containing acidic

sulphated polysaccharides obtained from U. rigida,

and epoxi-activated Sepharose 6B (GE Healthcare), as

previously described (Castro et al., 2008). Kidney

leucocyte lysates were applied to a column

(1 cm � 8 cm) in 50 mM Tris–HCl, pH 7.5, and eluted

with the same buffer containing different amounts of

NaCl (between 0.5 and 1.0 M NaCl). Eluted fractions

were combined, dialyzed against 20 mM sodium

phosphate buffer pH 7.0, concentrated by Amicon

and lyophilized.

2.6. Peroxidase activity

Enzyme assay peroxidase activity was measured by

spectrophotometry, with the chromogen ortho-pheny-

lenediamine (OPD, Sigma–Aldrich), as previously

described (Castro et al., 2008). Purified MPO and KL

lysates diluted in a solution of OPD (0.4 mg/ml in

0.05 M phosphate–citrate buffer, pH 5.0) were incu-

bated with H2O2 (0.4 ml/ml of commercial solution).

After 30 min incubation at room temperature, the

reaction was stopped by adding 3N sulphuric acid, and

the absorbance was determined at 492 nm. In order to

determine whether the RESV had a reversible or

irreversible inhibitory effect, the purified MPO was

incubated for 1 h with RESVat 100 mM and the enzyme

was then separated from the polyphenol by filtration in a

Microcon YM-10 Centrifugal Filter Unit (Millipore).

Filtration units were centrifuged at 10,000 � g for

10 min, the filtrate was then discarded and the enzyme

retained on the membrane was redissolved in PBS to the

original volume and the enzymatic activity determined

as described above.

2.7. Migration assay

Kidney leucocytes (2 � 105 cells) in L-15 medium

with no FCS, but containing 0.2 mg/ml BSA (Sigma–

Aldrich) were seeded in the upper chamber of a tissue

culture plate well insert with PCF membrane of pore

diameter 3 mm (Tissue culture-treated IsosporeTM

track-etched PVP-free polycarbonate; Millipore,

USA). The lower chamber was filled with 1 ml of L-

15 medium containing 2 mg/ml BSA and the stimulus,

i.e., ascitic fluid or P. dicentrarchi proteases. After

R. Castro et al. / Veterinary Immunology and Immunopathology 126 (2008) 9–19 11

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incubation of plates for 24 h at 18 8C, migrated cells

were scraped from the lower surface of the membrane

with a cell scraper (Nunc) and then suspended in the

medium in the lower chamber and all migrating cells

(both adherent and those in suspensions) were counted

in a haemocytometer.

2.8. Respiratory burst activity

Extracellular reactive oxygen species produced in

the respiratory burst assays were quantified by the use of

the fluorogenic reagent OxyBURST Green dihydro-

20,4,5,6,7,70-hexafluorofluorescein/BSA (Molecular

Probes), as previously described (Leiro et al.,

2004a,b). Assays were performed in 96-well micro-

culture plates by adding 100 ml of a suspension

containing 106 KL/ml of DMEM medium without

phenol red (Sigma–Aldrich). Cells were then incubated

with 10 mg/ml of fluorescent probe for 2 min at 23 8Cand stimulated by addition of 1 mg/ml of PMA. In some

assays, the medium contained RESV or L(+) ascorbic

acid. Fluorescence emission was then determined in a

microplate fluorescent reader (Bio-Tek instrument;

excitation at 488 nm, detection 530 nm, 55 min). The

results are expressed as increase in fluorescence

(arbitrary units) per minute.

2.9. Assay for measuring extracellular MPO

activity

The activity of extracellular MPO released by

incubation of turbot KL with PMA (100 ng/ml) was

determined in 96-well flat bottom microtitre plates. Test

wells received 100 ml of 106 KL incubated with 0.1 mg/

ml of PMA and RESV (100, 50 and 25 mM) in HBSS.

Control wells received only PMA and DMSO (10 mM)

and plates were incubated for 1 h at 21 8C. After

incubation, the plates were centrifuged at 600 � g for

5 min, and 89 ml of supernatant from each well was

transferred to another plate and mixed with 10 ml of a

solution of OPD (4 mg/ml in 0.05 M phosphate–citrate

buffer, pH 5.0) and 1 ml of H2O2 at 0.5%. After 30 min

incubation at room temperature, the reaction was

stopped by adding 25 ml of 3N sulphuric acid, and

the absorbance was determined at 492 nm.

2.10. Intracellular MPO activity

The intracellular MPO activity was visualized

microscopically as previously described (Leiro et al.,

2000). Kidney leucocyte cells were incubated with

RESV (100, 50 or 25 mM) for 1 h at 21 8C. After

incubation, cell suspensions were smeared on micro-

scope slides, fixed for 1 min in PBS containing 1%

formaldehyde and 2% glutaraldehyde, and the MPO

activity was determined by incubating the cells for

15 min with 0.02% of 3,30-diaminobenzidine tetrahy-

drochloride (DAB) and 0.006% H2O2 in 0.15 M Tris

buffer pH 7.6. The slides were then washed three times

with Tris buffer and cells were stained with haemotoxylin

for 4 min. After dehydration with ethanol, permanent

mounts were prepared with Eukitt. Stained cells (dark

brown cytoplasm) were counted as peroxidase-positive.

2.11. Determination of PGE2 in culture

supernatants

Aliquots (100 ml) of 107 KL/ml were incubated for

24 h at 18 8C with 100 mg/ml of ASP in the presence or

absence of indomethacin (10 mg/ml), a non-selective

inhibitor of cyclooxygenase-2-enzymatic activity, in 96-

well microculture plates (Leiro et al., 2007). The plates

were centrifuged at 400� g for 5 min, and prostaglandin

E2 (PGE2) levels were determined in the supernatant by a

competitive binding immunoassay (R&D Systems), by

following the manufacturer’s instructions. Briefly,

100 ml of PGE2 standard (dilutions from 1000 to

7.8 pg/ml in assay buffer containing blocking proteins

and preservative, buffer ED1) or samples (in assay buffer)

were incubated for 18 h at 4 8C with 50 ml of PGE2

conjugated to alkaline phosphatase (PGE2HS) and 50 ml

of mouse monoclonal antibody against PGE2. The wells

were washed three times with wash buffer, then 200 ml of

p-nitrophenylphosphate ( pNPP) in buffered solution

were added to each well, the plates were incubated for 1 h

at 37 8C and then the reaction stopped with 50 ml of

trisodium phosphate (TSP) solution. The optical density

(OD) was determined in a microplate reader (Titertek

Multiscan, Flow Laboratories) at 405 nm, with wave-

length correction between 570 and 590 nm. To estimate

the concentrations of PGE2 in samples, a calibration

curve of PGE2 concentration against 100B/B0 was

constructed (B: average OD for that concentration minus

non-specific binding; B0: average OD in wells without

PGE2HS minus non-specific binding; non-specific

binding: average OD in wells without either PGE2HS

or anti-PGE2 antibody).

2.12. Isolation of RNA, production of cDNA and

reverse transcriptase-polymerase chain reaction

(RT-PCR)

Total RNA without any contaminating DNA from

KL samples (107 cells/ml) was isolated with an mRNA

R. Castro et al. / Veterinary Immunology and Immunopathology 126 (2008) 9–1912

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isolation kit (NucleoSpin1; Macherey-Nagel), in

accordance with the manufacturer’s instructions. The

resulting RNA was dried and dissolved in diethylpyr-

ocarbonate (DEPC)-treated RNase-free water at a

concentration of 1 mg/ml. cDNA synthesis (25 ml/

reaction) was achieved with 1.25 mM random hexamer

primers (Roche) as described previously (Leiro et al.,

2003). One microlitre of cDNA was amplified by PCR

with the MPO forward/reverse primer pair, 50-CAA-

GAGAGAGCAGATCAACGC-30/50-GGCTGGATGG-

CCAAGTGG-30 (GenBank of NCBI accession number

EF112175) and turbot b-actin forward/reverse primer

pair, 50-AACTGGGATGACATGGAGA-30/50-GTCAG-

GATCTTCATGAGGTA-30 (NCBI, accession number

AY0083055) as a control for gene expression (Castro

et al., 2008). These primers amplified fragments of

567 bp (MPO) and 330 bp (b-actin). Thermal cycling

conditions were as follows: initial denaturating at 94 8Cfor 5 min; 30 cycles at 94 8C for 30 s, 65 8C (MPO) or

52 8C (b-actin) for 45 s, and 72 8C for 1 min and finally,

a 7-min extension phase at 72 8C. In all experiments,

RT-PCR controls (without RNA or without RT) were

included; no amplification products were obtained in

any of the experiments. Aliquots (20 ml) of PCR

products were separated on a 2% agarose gel, stained

with ethidium bromide and photographed with a digital

camera. Bands were quantified in tagged image file

format (TIFF) with densitometry analysis software

(ImageMaster Total Laboratory, version 2.00, GE

Healthcare).

2.13. Statistical analysis

Results shown are expressed as means � standard

error of the mean (S.E.M.). Statistical significances

(a = 0.05) were determined by one-way analysis of

variance (ANOVA) followed by Tukey–Kramer test for

multiple comparisons.

3. Results

3.1. Effect of RESV on migration of turbot kidney

leucocytes

The effect of RESV on migration of turbot KL

stimulated with ascitic fluid obtained from turbot

infected experimentally with the pathogen ciliate P.

dicentrarchi was evaluated, and the results obtained

after adding RESV or parasite proteases to the fluid are

shown in Fig. 1. At concentrations higher than 25 mM,

RESV had a significant inhibitory effect on migration of

KL stimulated with ascitic fluid. This inhibition was

equivalent to that obtained with cysteine proteases

obtained from P. dicentrarchi (Fig. 1).

3.2. Effect of RESV on respiratory burst of turbot

kidney leucocytes stimulated with phorbol myristate

acetate (PMA)

The KL stimulated with PMA displayed a highly

significant increase in the extracellular production of

ROS. However, the levels of extracellular ROS

decreased significantly when cells were incubated with

RESV at concentrations higher than 25 mM. This

decrease was equivalent to that induced by L(+)

ascorbic acid, a scavenger of ROS, at 1 mM (Fig. 2).

3.3. Effect of RESV on PGE2 production by kidney

leucocytes stimulated with acidic sulphated

polysaccharides (ASPs)

The addition of ASP from the seaweed U. rigida

(50 mg/ml) caused an increase in the levels of PGE2 in

the supernatants of cultures of turbot KL (Fig. 3). At

concentrations higher than 25 mM, RESV significantly

inhibited the production of PGE2 (Fig. 3).

3.4. Effect of RESV on the intracellular and

extracellular activity of turbot kidney leucocyte

MPO

Only the neutrophil precursors and the neutrophils

showed intracellular peroxidase activity as determined

in kidney leucocytes by the use of DAB as chromogen

R. Castro et al. / Veterinary Immunology and Immunopathology 126 (2008) 9–19 13

Fig. 1. Migration activity of turbot kidney leucocytes (KLs) in

response to ascitic fluid from turbot infected experimentally with

Philasterides dicentrarchi and DMSO (10 mM) (control group), and to

ascitic fluid containing resveratrol (RESV) or P. dicentrarchi cysteine

proteases. Values are means � standard errors. Asterisks indicate

statistically significant differences (P < 0.01; n = 5) relative to the

control.

Author's personal copy

and H2O2 as substrate (Fig. 4A). Interestingly, a

noteworthy decrease in intracellular peroxidase activity

was observed after addition of RESV to the cells, and this

effect was more evident at higher doses of the polyphenol

(Fig. 4A). The addition of PMA (100 ng/ml) to KL

caused exocytosis of MPO to the culture medium

(Fig. 4B). However, the extracellular peroxidase activity

in the cell culture supernatants was strongly inhibited by

RESV at concentrations higher than 25 mM (Fig. 4B).

3.5. Effect of RESV on enzymatic activity of turbot

neutrophil myeloperoxidase (MPO)

RESV caused significant inhibition of the peroxidase

activity of KL lysates (Fig. 5A). Turbot MPO was

purified and the effects of RESV on the activity of this

enzyme determined. It was found that the activity was

greatly inhibited when MPO was incubated with the

polyphenol (Fig. 5B). The effects of RESV on the

velocity of the reaction were also determined; RESV

clearly decreased the maximal velocity of the enzymatic

reaction generated by the MPO (Fig. 5C). In addition,

the enzymatic activity was recovered when RESV was

eliminated (Fig. 5D).

3.6. Effect of RESV on MPO expression

A semi-quantitative RT-PCR was used to assess the

effect of different concentrations of RESV (25, 50 and

100 mM) on MPO mRNA levels in turbot KL (Fig. 6).

Kidney lecuocytes were stimulated with PMA and

RESV for 1 h. At 25–50 mM, RESV downregulated

turbot MPO mRNA levels and, at 100 mM, it

completely suppressed MPO gene expression (Fig. 6B).

4. Discussion

Resveratrol, a polyphenolic phytoalexin, shows a

wide ranging biological activity and tremendous

clinical potential in human medicine because of its

positive effects on a variety of disease states, including

anti-thrombogenic, anti-inflammatory, cardio-protec-

tive, neuro-protective, anti-aging, and cancer preventive

and therapeutic activities (Holme and Pervaiz, 2007).

The innate immune response is considered of vital

importance for disease resistance in fish, particularly in

view of the generally sluggish acquired immune

response and late ontogenic appearance of acquired

parameters in many fish species (Magnadottir, 2006).

Host resistance against pathogens depends on the

performance of the first line of defence of the innate

immune system, including activation of neutrophils,

tissue macrophages, monocytes, dendritic cells, eosi-

nophils and natural killer (NK) cells (Papazahariadou

et al., 2007).

The migration of leucocytes to the site of injury is an

important step in the inflammatory process (Zabel et al.,

2006; Mathias et al., 2006). In the present study it was

found that RESV caused significant inhibition of the

migratory response of turbot KL to peritoneal fluid

generated after infection of the fish with the scutico-

ciliate parasite P. dicentrarchi. This inhibition was

R. Castro et al. / Veterinary Immunology and Immunopathology 126 (2008) 9–1914

Fig. 2. Extracellular production of reactive oxygen species (ROS),

measured by the use of the fluorescent reagent OxyBURST Green

H2HFF BSA, of turbot kidney leucocytes incubated for 1 h with 1 mg/

ml of phorbol myristate acetate (PMA). In some assays, the medium

contained resveratrol (RESV) or 1 mM of L(+) ascorbic acid. Values

shown are means � standard errors (n = 5) of increase in fluorescence

(in arbitrary units). Asterisks indicate statistically significant differ-

ences (P < 0.01) relative to the control group stimulated with PMA

and DMSO (10 mM).

Fig. 3. Production of prostaglandin E2 (PGE2) in 24 h supernatants

from turbot kidney leucocytes stimulated with 100 mg/ml of acidic

sulphated polysaccharides (ASPs) from the seaweed Ulva rigida and/

or resveratrol (RESV) and indomethacin (INDO), a non-selective

inhibitor of COX enzymatic activity. Values shown are means � stan-

standard errors (n = 5). Asterisks indicate statistically significant

differences (P < 0.01) relative to the control stimulated with ASP

and 10 mM DMSO.

Author's personal copy

similar to that induced by the parasite proteases, which,

as previously demonstrated, inhibit the migration of

turbot KL (Castro et al., 2004, 2006). In mammals, RESV

also inhibits chemotactic and calcium mobilization

responses of phagocytic cells to selected chemoattrac-

tants, which suggests that the inhibition of the function of

chemoattractant receptors may contribute to the anti-

inflammatory properties of RESV (Tao et al., 2004).

In a previous study it was found that turbot

phagocytes, especially neutrophils, produce large

amounts of ROS after stimulation (Castro et al.,

1999; Couso et al., 2001). In the present study, RESV

R. Castro et al. / Veterinary Immunology and Immunopathology 126 (2008) 9–19 15

Fig. 4. Intracellular and extracellular enzymatic peroxidase activity by turbot kidney leucocytes (KLs). (A) Light micrograph showing kidney

neutrophils incubated for 1 h with resveratrol (RESV) and then incubated with the chromogen 3,30-diaminobenzidine tetrahydrochloride (DAB). (B)

Extracellular peroxidase activity, determined with the chromogen ortho-phenylenediamine (OPD), in supernatants obtained from turbot kidney

leucocytes stimulated for 1 h with 100 ng/ml of phorbol myristate acetate (PMA) and/or resveratrol (RESV). Values shown in bars are

means � standard errors (n = 5). Asterisks indicate significant differences (P < 0.01) relative to the groups indicated in brackets and numbering

is indicated at the base of bar.

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caused a significant reduction in the production of ROS

induced by PMA in turbot KL, at levels comparable to

those of the reference antioxidant L(+) ascorbic acid.

RESValso inhibits the production of ROS in neutrophils

and macrophages of mammalian species, including

humans (Leiro et al., 2002; Ignatowicz et al., 2003;

Cavallaro et al., 2003; Kohnen et al., 2007). The

production of ROS during the respiratory burst is

intended to kill invading pathogens (Valko et al., 2007)

but ROS also induces oxidative stress and free radical-

induced injuries, which has detrimental effects on parts

of the organism such as the nervous system (Reynolds

et al., 2007). The decrease in the amount of ROS present

in turbot leucocytes incubated with RESV may be

related to a scavenging effect or to inhibition of internal

production. Cavallaro et al. (2003) have suggested that

RESV enters the neutrophils and inhibits ROS produc-

tion, although the mechanism has not yet been

established. Although the activity of RESV as a

scavenger of ROS was not investigated in the present

study, it has previously been demonstrated that RESV

was ineffective at scavenging superoxide anions (O2�)

generated enzymatically by a hypoxanthine/xanthine

oxidase (HX/XO) system (Orallo et al., 2002).

One of the possible ways that RESV provides

protection is by down regulation of the inflammatory

responses, including the inhibition of synthesis and

release of pro-inflammatory mediators such as the

eicosanoid PGE2 produced by monocytes and throm-

bocytes from arachidonic acid (Das and Das, 2007). In

the present study, the effects of RESVon the production

of PGE2 by kidney leucocytes stimulated with seaweed

R. Castro et al. / Veterinary Immunology and Immunopathology 126 (2008) 9–1916

Fig. 5. Effect of resveratrol (RESV) on turbot myeloperoxidase (MPO) enzymatic activity. (A) Total peroxidase activity in lysates of kidney

leucocytes (KL) measured with ortho-phenylenediamine (OPD) as chromogen. Resveratrol was included in the reaction mixture. (B) Activity of

purified turbot MPO measured by the use of OPD. Resveratrol was included in the reaction mixture. (C) Simple Michaelis–Menten kinetics of MPO

showing the velocity of reaction (V, absorbance at 492 nm in 20 min) plotted against substrate concentration (S, H2O2 in percentage). The effect of

RESV at 0 (control), 25, 50 and 100 mM on the kinetics is included. (D) MPO activity in presence of 0 or 100 mM RESV (none) and the activity of

MPO preincubated with RESVand separated from the polyphenol by filtration (filtered). Values shown are means � standard errors (n = 5). Asterisk

indicates statistically significant differences (P < 0.01) relative to the control.

Author's personal copy

polysaccharides, which stimulate the production of

prostaglandins in these cells (Castro et al., 2006), were

investigated and it was found that this polyphenol also

inhibited the production of PGE2. In mammals, RESV

has a suppressive effect on prostaglandin biosynthesis,

and its action on the arachidonic acid mobilization and

on cyclooxygenase 2 (COX-2) induction has been

established (Martinez and Moreno, 2000; Leiro et al.,

2004a, b).

The neutrophils of most fish species, including

turbot, contain abundant amounts of MPO. As men-

tioned earlier, neutrophils and their precursors are the

only kidney leucocytes that show MPO activity in turbot

and the present results demonstrate that RESV inhibits

the activity of this enzyme. RESV also inhibits MPO

activity in neutrophils of humans and other mammals

(Cavallaro et al., 2003; Kohnen et al., 2007). Kohnen

et al. (2007) suggested that RESV acts as a competitive

substrate for MPO and also inhibits the activity by direct

interaction with the enzyme, mainly affecting the MPO

released to the extracellular milieu. In the present study,

RESV inhibited extracellular and also intracellular

MPO, which indicates that this molecule can cross the

cell membrane. MPO uses hydrogen peroxide to

oxidase substrates that may be toxic to microorganisms,

and a close connection between MPO and metabolism

of NO (another important mediator of inflammation)

has been described, which suggests that MPO also acts

as an NO oxidase (Eiserich et al., 2002). However, the

results of recent studies also indicate that this enzyme

may be involved in the regulation of cellular home-

ostasis and may play a central role in initiation and

propagation of acute and chronic vascular inflammatory

disease (Lau and Baldus, 2006). This contribution to the

inflammatory response may have deleterious effects on

the organism. The gene that codifies turbot MPO has

been isolated and partially characterized (Castro et al.,

2008), and in the present study it was demonstrated for

the first time that the RESV inhibits MPO expression in

this fish species in a dose-dependent way, indicating that

the polyphenol blocks expression of the gene that

codifies this enzyme, in addition to the enzymatic

activity. The results obtained in the present study

indicate that RESV acts as a general inhibitor of

peroxidase activity and that this phenomenon may also

be related to the inhibition observed in PGE2 synthesis

through inhibition of the activity of prostaglandin

endoperoxidase H synthase (PGHS), a key enzyme in

the formation of PGs from arachidonic acid (Marcin-

kiewicz, 2003).

In summary, the present results indicate that RESV

elicits important modulation of turbot leucocyte

activity, thereby blocking events involved in the

inflammatory response, such as cell migration, respira-

tory burst activity, and PG synthesis. In addition, RESV

behaved as a powerful inhibitor of the activity and

expression of fish MPO. To our knowledge, this is the

first published report of modulation of fish leucocyte

response by a polyphenol of natural origin.

Acknowledgements

This work was financially supported by Spanish

Grants AGL2006-12872-C02-01/ACU and AGL2004-

01896 from the Comision Interministerial de Ciencia y

Tecnologıa (CICYT), FISS PI061537 from the Minis-

terio de Sanidad y Consumo, CSD2007-00002, Aqua-

genomics project funded through the Consolider-Ingenio

2010 programme, by the Spanish Ministerio de Educa-

cion y Ciencia (MEC), INCITE07PXI203039ES and

PGIDT5BTF20302PR from the Xunta de Galicia.

Francisco Orallo is especially grateful to the

Consellerıa de Educacion e Ordenacion Universitaria

R. Castro et al. / Veterinary Immunology and Immunopathology 126 (2008) 9–19 17

Fig. 6. Effect of resveratrol (RESV) on myeloperoxidase (MPO)

mRNA levels in turbot kidney leucocytes, as determined by RT-

PCR. Cells were incubated for 1 h with PMA and/or resveratrol.

(A) Agarose analysis (2%) of RT-PCR products with primers for MPO

(567 bp) or the housekeeping gene b-actin (330 bp). (B) Quantifica-

tion of normalized RT-PCR products of MPO, by densitometry

(optical density (OD) of MPO band/OD of b-actin band). Values

are means � standard errors (n = 5) and asterisks indicate statistically

significant differences (P < 0.01) relative to the control stimulated

with PMA and DMSO. Mw: molecular weight markers (100 bp

ladder).

Author's personal copy

de la Xunta de Galicia (Spain) for granting him

financial support for intensification of research activity

and reduction of teaching activity during the academic

year 2007–2008 [Programa de promocion de intensi-

ficacion de la actividad investigadora en el sistema

Universitario de Galicia (SUG)].

References

Castro, R., Couso, N., Obach, A., Lamas, J., 1999. Effect of different

b-glucans on the respiratory burst of turbot (Psetta maxima) and

gilthead seabream (Sparus aurata) phagocytes. Fish Shellfish

Immunol. 9, 529–541.

Castro, R., Piazzon, M.C., Noya, M., Leiro, J.M., Lamas, J., 2008.

Isolation and molecular cloning of a fish myeloperoxidase. Mol.

Immunol. 45, 428–437.

Castro, R., Piazzon, M.C., Zarra, I., Leiro, J., Noya, M., Lamas, J.,

2004. Stimulation of turbot phagocytes by Ulva rigida C. Agardh

polysaccharides. Aquaculture 254, 9–20.

Castro, R., Zarra, I., Lamas, J., 2006. Water-soluble seaweed extracts

modulate the respiratory burst activity of turbot phagocytes.

Aquaculture 229, 67–78.

Cavallaro, A., Ainis, T., Bottari, C., Fimiani, V., 2003. Effect of

resveratrol on some activities of isolated and in whole blood

human neutrophils. Physiol. Res. 52, 555–562.

Chen, Y., Tseng, S.H., 2007. Review. Pro- and anti-angiogenesis

effects of resveratrol. In Vivo 21, 365–370.

Collazos, M.E., Barriga, C., Ortega, E., 1995. Effect of high summer

temperatures upon granulocyte phagocytic function of the tench

(Tinca tinca L.). Comp. Immunol. Microbiol. Infect. Dis. 18,

115–121.

Couso, N., Castro, R., Noya, M., Lamas, J., 2001. Location of

superoxide production sites in turbot neutrophils and gilthead

seabream acidophilic granulocytes during phagocytosis of glucan

particles. Dev. Comp. Immunol. 25, 607–618.

Das, S., Das, D.K., 2007. Anti-inflammatory responses of resveratrol.

Inflamm. Allergy Drug Targets 6, 168–173.

Eiserich, J.P., Baldus, S., Brennan, M.L., Ma, W., Zhang, C., Tousson,

A., Castro, L., Lusis, A.J., Nauseef, W.M., White, C.R., Freeman,

B.A., 2002. Myeloperoxidase, a leukocyte-derived vascular NO

oxidase. Science 296, 2391–2394.

Fraga, C.G., 2007. Plant polyphenols: how to translate their in vitro

antioxidant actions to in vivo conditions. IUBMB Life 59,

308–315.

Holme, A.L., Pervaiz, S., 2007. Resveratrol in cell fate decisions. J.

Bioenerg. Biomembr. 39, 59–63.

Iglesias, R., Parama, A., Alvarez, M.F., Leiro, J., Fernandez, J.,

Sanmartın, M.L., 2001. Philasterides dicentrarchi (Ciliophora,

Scuticociliatida) as the causative agent of scuticociliatosis in

farmed turbot Scophthalmus maximus in Galicia (NW Spain).

Dis. Aquat. Org. 46, 47–55.

Iglesias, R., Parama, A., Alvarez, M.F., Leiro, J., Ubeira, F.M.,

Sanmartın, M.L., 2003. Philasterides dicentrarchi (Ciliophora:S-

cuticociliatida) expresses surface immobilization antigens that

probably induce protective immune responses in turbot. Parasitol-

ogy 126, 125–134.

Ignatowicz, E., Balana, B., Vulimiri, S.V., Szaefer, H., Baer-Dubowska,

W., 2003. The effect of plant phenolics on the formation of 7,12-

dimethylbenz[a]anthracene–DNA adducts and TPA-stimulated

polymorphonuclear neutrophils chemiluminescence in vitro. Tox-

icology 189, 199–209.

Khanna, D., Sethi, G., Ahn, K.S., Pandey, M.K., Kunnumakkara, A.B.,

Sung, B., Aggarwal, A., Aggarwal, B.B., 2007. Natural products

as a gold mine for arthritis treatment. Curr. Opin. Pharmacol. 7,

344–351.

Kohnen, S., Franck, T., Van Antwerpen, P., Boudjeltia, K.Z.,

Mouithys-Mickalad, A., Deby, C., Moguilevsky, N., Deby-

Dupont, G., Lamy, M., Serteyn, D., 2007. Resveratrol inhibits

the activity of equine neutrophil myeloperoxidase by a direct

interaction with the enzyme. J. Agric. Food Chem. 55, 8080–8087.

Kumar, V., Abbas, A.K., Fausto, N., 2004. Robbins and Cotran

Pathologic Basis of Disease. Elsevier Saunders, Philadelphia, PA.

Kundu, J.K., Shin, Y.K., Surh, Y.J., 2004. Resveratrol modulates

phorbol ester-induced pro-inflammatory signal transduction path-

ways in mouse skin in vivo, NF-kappaB and AP-1 as prime targets.

Biochem. Pharmacol. 72, 1506–1515.

Lau, D., Baldus, S., 2006. Myeloperoxidase and its contributory role

in inflammatory vascular disease. Pharmacol. Ther. 111, 16–26.

Leiro, J., Alvarez, E., Arranz, J.A., Laguna, R., Uriarte, E., Orallo, F.,

2004a. Effects of cis-resveratrol on inflammatory murine macro-

phages: antioxidant activity and down-regulation of inflammatory

genes. J. Leukoc. Biol. 75, 1156–1165.

Leiro, J., Garcıa, D., Arranz, J.A., Delgado, R., Sanmartın, M.L.,

Orallo, F., 2004b. An Anacardiaceae preparation reduces the

expression of inflammation-related genes in murine macrophages.

Int. Immunopharmacol. 4, 991–1003.

Leiro, J., Alvarez, E., Garcıa, D., Orallo, F., 2002. Resveratrol

modulates rat macrophage functions. Int. Immunopharmacol. 2,

767–774.

Leiro, J., Arranz, J.A., Fraiz, N., Sanmartın, M.L., Quezada, E., Orallo,

F., 2005. Effect of cis-resveratrol on genes involved in nuclear

factor kappa B signalling. Int. Immunopharmacol. 5, 393–406.

Leiro, J., Castro, R., Arranz, J.A., Lamas, J., 2007. Immunomodulat-

ing activities of acidic sulphated polysaccharides obtained from

the seaweed Ulva rigida C. Agardh. Int. Immunopharmacol. 7,

879–888.

Leiro, J., Ortega, M., Sanmartın, M.L., Ubeira, F.M., 2000. Non-

specific responses of turbot (Scophthalmus maximus L.) adherent

cells to microsporidian spores. Vet. Immunol. Immunopathol. 75,

81–95.

Leiro, J.M., Alvarez, 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-beta genes. Biochem.

Pharmacol. 65, 1361–1371.

Magnadottir, B., 2006. Innate immunity of fish (overview). Fish

Shellfish Immunol. 20, 137–151.

Marcinkiewicz, J., 2003. Prostanoids and MPO–halide system pro-

ducts as a link between innate and adaptive immunity. Immunol.

Lett. 89, 187–191.

Martinez, J., Moreno, J.J., 2000. Effect of resveratrol, a natural

polyphenolic compound, on reactive oxygen species and prosta-

glandin production. Biochem. Pharmacol. 59, 865–870.

Mathias, J.R., Perrin, B.J., Liu, T.X., Kanki, J., Look, A.T., Hutten-

locher, A., 2006. Resolution of inflammation by retrograde che-

motaxis of neutrophils in transgenic zebrafish. J. Leukoc. Biol. 80,

1281–1288.

Nam, N.H., 2006. Naturally occurring NF-kappaB inhibitors. Mini

Rev. Med. Chem. 6, 945–951.

Orallo, F., Alvarez, E., Camina, M., Leiro, J.M., Gomez, E., Fernan-

dez, P., 2002. The possible implication of trans-resveratrol in the

R. Castro et al. / Veterinary Immunology and Immunopathology 126 (2008) 9–1918

Author's personal copy

cardioprotective effects of long-term moderate wine consumption.

Mol. Pharmacol. 61, 294–302.

Papazahariadou, M., Athanasiadis, G.I., Papadopoulos, E., Symeoni-

dou, I., Hatzistilianou, M., Castellani, M.L., Bhattacharya, K.,

Shanmugham, L.N., Conti, P., Frydas, S., 2007. Involvement of

NK cells against tumors and parasites. Int. J. Biol. Markers 22,

144–153.

Parama, A., Castro, R., Arranz, J.A., Sanmartın, M.L., Lamas, J.,

Leiro, J., 2007. Scuticociliate cysteine proteinases modulate turbot

leucocyte functions. Fish Shellfish Immunol. 23, 945–956.

Parama, A., Iglesias, R., Alvarez, M.F., Leiro, J., Ubeira, F.M.,

Sanmartın, M.L., 2004a. Cysteine proteinase activities in the fish

pathogen Philasterides dicentrarchi (Ciliophora: Scuticocilia-

tida). Parasitology 128, 541–548.

Parama, A., Iglesias, R., Alvarez, M.F., Sanmartın, M.L., Leiro, J.,

2004b. Chemotactic responses of the fish-parasitic scuticociliate

Philasterides dicentrarchi to blood and blood components of the

turbot Scophthalmus maximus, evaluated using a new microplate

multiassay. J. Microbiol. Methods 58, 361–366.

Pedrera, I.M., Collazos, M.E., Ortega, E., Barriga, C., 1992. In vitro

study of the phagocytic processes in splenic granulocytes of the

tench (Tinca tinca L.). Dev. Comp. Immunol. 16, 431–439.

Plouffe, D.A., Hanington, P.C., Walsh, J.G., Wilson, E.C., Belosevic,

M., 2005. Comparison of select innate immune mechanisms of fish

and mammals. Xenotransplantation 12, 266–277.

Reynolds, A., Laurie, C., Mosley, R.L., Gendelman, H.E., 2007.

Oxidative stress and the pathogenesis of neurodegenerative dis-

orders. Int. Rev. Neurobiol. 82, 297–325.

Stakauskas, R., Schuberth, H.J., Leibold, W., Steinhagen, D., 2007.

Modulation of carp (Cyprinus carpio) neutrophil functions during

an infection with the haemoparasite Trypanoplasma borreli. Fish

Shellfish Immunol. 23, 446–458.

Tao, H.Y., Wu, C.F., Zhou, Y., Gong, W.H., Zhang, X., Iribarren, P.,

Zhao, Y.Q., Le, Y.Y., Wang, J.M., 2004. The grape component

resveratrol interferes with the function of chemoattractant

receptors on phagocytic leukocytes. Cell. Mol. Immunol. 1,

50–56.

Valenzano, D.R., Cellerino, A., 2006. Resveratrol and the pharmacol-

ogy of aging: a new vertebrate model to validate an old molecule.

Cell Cycle 5, 1027–1032.

Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T., Mazur, M.,

Telser, J., 2007. Free radicals and antioxidants in normal phy-

siological functions and human disease. Int. J. Biochem. Cell

Biol. 39, 44–84.

Whyte, S.K., 2007. The innate immune response of finfish—a review

of current knowledge. Fish Shellfish Immunol. 23, 1127–1151.

Zabel, B.A., Zuniga, L., Ohyama, T., Allen, S.J., Cichy, J., Handel,

T.M., Butcher, E.C., 2006. Chemoattractants, extracellular pro-

teases, and the integrated host defense response. Exp. Hematol. 34,

1021–1032.

R. Castro et al. / Veterinary Immunology and Immunopathology 126 (2008) 9–19 19