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Immunomodulating activities of acidic sulphated polysaccharidesobtained from the seaweed Ulva rigida C. Agardh

José M. Leiro a, Rosario Castro b, Jon A. Arranz a, Jesús Lamas b,⁎

a Laboratorio de Parasitología, Instituto de Investigación y Análisis Alimentarios, Universidad de Santiago de Compostela,15782 Santiago de Compostela, Spain

b Departamento de Biología Celular y Ecología; Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain

Received 6 November 2006; received in revised form 16 February 2007; accepted 16 February 2007

Abstract

Water-soluble acidic polysaccharides from the cell walls of Ulva rigida are mainly composed of disaccharides that containglucuronic acid and sulphated rhamnose. The structure of disaccharides resembles that of glycosaminoglycans (GAGs) as they bothcontain glucuronic acid and sulphated sugars. Glycosaminoglycans occur in the extracellular matrix of animal connective tissuesbut can also be produced by leucocytes at inflammatory sites. Certain types of GAGs can even activate macrophages and thereforethe acidic polysaccharides from U. rigida probably modulate macrophage activity. In the present study, we evaluated the effects ofU. rigida polysaccharides on several RAW264.7 murine macrophage activities, including expression of inflammatory cytokinesand receptors, nitric oxide and prostaglandin E2 (PGE2) production, and nitric oxide synthase 2 (NOS-2) and cyclooxygenase-2(COX-2) gene expression. U. rigida acidic polysaccharides induced a more than two-fold increase in the expression of severalchemokines (chemokine (C motif) ligand 1, chemokine (C-X-C motif) ligand 12, chemokine (C-C motif) ligand 22 and chemokine(C-X-C motif) ligand 14 (Cxcl14)) and in the expression of IL6 signal transducer and IL12 receptor beta 1. Incubation ofmacrophages with U. rigida polysaccharides also induced an increase in nitrite production, although this effect decreasedconsiderably after desulphation of polysaccharides, suggesting that the sulphate group is important for the stimulatory capacity ofthese molecules. U. rigida polysaccharides also stimulated macrophage secretion of PGE2 and induced an increase in COX-2 andNOS-2 expression. The results indicate that U. rigida acid polysaccharide can be used as an experimental immunostimulant foranalysing inflammatory responses related to macrophage functions. In addition, these polysaccharides may also be of clinicalinterest for modifying certain macrophage activities in diseases where macrophage function is impaired or needs to be boosted.© 2007 Elsevier B.V. All rights reserved.

Keywords: RAW264.7 macrophages; Ulva rigida; Sulphated polysaccharides; Cytokine expression; Nitric oxide; Prostaglandins

1. Introduction

Macrophages are multifunctional cells that have acentral role in the innate and adaptive immune responses[1]. Macrophages protect the host by engulfing andkilling pathogens, present antigens to lymphocytes andrelease numerous biologically active molecules that

International Immunopharmacology 7 (2007) 879–888www.elsevier.com/locate/intimp

⁎ Corresponding author. Departamento de Biología Celular yEcología, Facultad de Biología, Rúa Lope Gómez de Marzoa s/n,15782 Santiago de Compostela, Spain. Tel.: +34 981563100x13376;fax: +34 981596004.

E-mail address: bfsuso@usc.es (J. Lamas).

1567-5769/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.intimp.2007.02.007

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regulate the activity of other cells. They participate ininflammatory processes, which are important in remov-ing pathogens and, depending on the stimuli, they mayalso participate in the resolution of inflammation byreleasing anti-inflammatory cytokines and eliminatingcell debris [2]. It is possible to modify the function ofmacrophages by inducing selective stimulation orsuppression of cell activity. Some substances such asbacterial lipopolysaccharides, fungal β-glucans orcertain types of carrageenans have been used to activatemacrophages [3–6]. However, other substances such assome carrageenans, dextran sulphate or fucoidan (amarine sulphated polysaccharide obtained from sea-weed) appear to inhibit macrophage functions [1,7].Both groups of substances may have biomedicalapplications and may be of potential use in stimulatingthe immune system or in controlling macrophage activityto reduce associated negative effects.

The cell walls of seaweeds are rich in matrixpolysaccharides of different shapes and with differentbiological properties. Some of those polysaccharidesappear to exert immunomodulatory activities in mammalsas they modify the activity of macrophages. Polysacchar-ides obtained from the two red algae Porphyra yezoensisand Gracilaria verrucosa stimulate phagocytosis andrespiratory burst in mouse macrophages in vitro and invivo [8–10]. Carrageenan, a sulphated polysaccharideobtained from red algae and known to be a potentinflammatory agent in rodents, primes mice leucocytes toproduce TNF-α in response to bacterial lipopolysaccha-ride [11]. Some types of carrageenans induce potentmacrophage activation [4,5,12]. However, other typesappear to impair macrophage functions, indicating theimportance of the molecular structure of these poly-saccharides in their immunomodulatory activities. Sea-weed polysaccharides also possess antitumour activity[13–15] and, in some cases, this activity was found to beassociated with macrophages, the presence of whichincreased the release of certain cytokines such as tumournecrosis factor-α (TNF-α) [8,16,17].

Ulva rigida is rich in cell-wall polysaccharides,including cellulose and water-soluble polysaccharidesthat contain sulphate groups. The main type of water-soluble polysaccharide is ulvan, the main component ofwhich is a disaccharide formed by β-D-glucuronic acid(1,4)-L-rhamnose 3 sulphate [18,19]. Interestingly, thisdisaccharide resembles glycosaminoglycans such ashyaluronan and chondroitin sulphate because they allcontain glucuronic acid and sulphate. Human mono-cytes and macrophages produce proteoglycans –mainlychondroitin sulphate proteoglycans – at inflammatorysites [20,21] and certain types of chondroitin sulphates

have been shown to activate monocytes and B-lymphocytes clones [22,23]. Furthermore, ulvan con-tains rhamnose, a sugar that is common in bacteria butnot in animal tissues. All of these structural character-istics make ulvan worthy of investigation. The aim ofthe present study was to determine if acidic sulphatedpolysaccharides obtained from the seaweed U. rigidamodulate the activity of murine macrophages.

2. Materials and methods

2.1. Preparation of the acid fractions from seaweedpolysaccharides

U. rigida C. Agardh was collected from the coast of Galicia(NW Spain). Seaweeds were homogenized with distilled water(25 g in 100 ml) in a dounce homogenizer and maintained for24 h at 4 °C. After centrifugation at 1500 ×g for 20 min, thesupernatants were collected and filtered (8 to 0.2 μm). Afterprecipitation of the proteins with 5% trichloroacetic acid(TCA) and dialysis of the supernatants against distilled water,polysaccharides were precipitated with ethanol and thenfractionated into neutral and acid sugars, as previouslydescribed [24,25]. Briefly, polysaccharides were fractionatedby anion exchange chromatography in a DEAE-Trisacrylcolumn (3 cm×30 cm) equilibrated with 5 mM phosphatebuffer (pH 7.0). Non-adsorbed and adsorbed fractions wererecovered, dialysed and concentrated to the initial volume byultrafiltration (membranes of NMWL 10,000 Da). Themolecular weight distribution of the acid fraction polymerswas determined by gel permeation chromatography on aSepharose CL-4B column (1.5 cm×90 cm), with blue dextran(2000 kDa, Sigma) and glucose (Sigma) as standards.

Protein contents were determined colorimetrically by theLowry method. Total sugar content of the extracts wasdetermined colorimetrically by the phenol-sulphuric method,with glucose as standard [26]. The uronic acid content wasdetermined by the m-hydroxydiphenyl method, with galacturo-nic acid as standard [27].

Polysaccharides were also analysed for monosaccharidecomposition by gas chromatography after 2N TFA hydrolysisand conversion of the hydrolysate into alditol acetatesfollowing a previously described method [28]. The alditolacetates obtained were analysed in a Hewlett Packard 5890 gaschromatograph system equipped with a SP-2330 capillarycolumn (30 m), at an oven temperature of 240 °C. Thetemperature of the injector and detector was 300 °C.

Desulphation of the acid fraction was carried out aspreviously described [24,25]. The extracts (10 ml) were passedthrough a Dowex 50W column (X-8, H+), and the effluent wasneutralized with pyridine and then lyophilised. The pyridiniumsalt obtained was desulphated with dimethyl sulphoxidecontaining 10% of distilled water. Aliquots of this solutionwere heated at 80 °C for 5 h. The reaction mixture was thendiluted with distilled water, dialysed against distilled water for4 days and concentrated to the initial volume to obtain the

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desulphated fraction. To compare the structures, the acid anddesulphated fractions were analysed by infrared spectroscopy,with a Bomem Michelson (Hartman & Braun) Fouriertransform-IR spectrophotometer (resolution 4 cm−1, andaveraging 64 scans/min) as previously described [24,25]. Todetermine the structure of the acidic sulphated polysaccharides,the samples were analysed by Proton Nuclear MagneticResonance (1H-NMR) spectroscopy. Solutions of oligosac-charides in D2O (0.6 mL, 99.9%; Sigma-Aldrich) wereanalysed at 25 °C with an Inova-750 NMR spectrometer anddiagnostic regions in the anomeric region of the spectraidentified as previously described [18], and chemical shiftswere measured with acetone at δ 2.225 as an internal reference.The NMR data were processed by use of the MestRe-Cprogram (bhttp://www.mestrec.comN, University of Santiago,Spain).

2.2. Cell culture

RAW264.7 murine macrophages (ATCC) were maintainedin Dulbecco's modified Eagle's medium with 4 mM L-glutamine, 10% foetal bovine serum (FBS), 100 U ml−1 ofpenicillin and 0.1 mg ml−1, in a CO2 incubator at 37 °C. Cellswere grown in culture flasks until 80–90% confluent and werethen incubated with the polysaccharides.

2.3. Macrophage RNA isolation

Macrophages (5×106 cells ml−1) were incubated withseveral concentrations of seaweed extracts for 24 h. After theincubation period, the culture medium was removed and cellswere washed twice with PBS. Isolation of total RNA withoutDNA contamination was carried out with an RNA isolation kit(NucleoSpin®; Macherey-Nagel, Germany) in accordance withthe manufacturer's instructions. To confirm the quality of theRNA, 10 μl of RNA was evaluated by denaturing formalde-hyde/agarose gel electrophoresis.

2.4. Nitric oxide production

Nitric oxide (NO) production was estimated on the basis ofnitrite production in macrophage culture supernatants, mea-sured by the Griess reaction, as described elsewhere [29].Briefly, cells were incubated for 48 h with several concentra-tions of sulphated and desulphated Ulva polysaccharides (0–100 μg ml−1) in culture medium, in the presence or absence ofinterferon-gamma (10 U ml−1), lipopolysaccharide (LPS)(100 ng ml−1) and 250 μM of the arginine analogue N(G)-monomethyl-L-arginine (L-NMMA). Aliquots (100 μl) wereremoved from the medium and mixed with an equal volume ofGriess reagent (1% sulphanilamide and 0.1% naphthylethyle-nediamine hydrochloride in 2.5% H3PO4). After incubation for10 min at room temperature, the absorbance was measured at530 nm in an ELISA reader (Titertek Multiscan, FlowLaboratories, Costa Mesa, USA). The concentration of nitritewas calculated with reference to a standard curve obtainedwith NaNO2 (1–200 μM in culture medium).

2.5. PGE2 production

The PGE2 levels in the macrophages supernatants weremeasured by a competitive binding immunoassay (R&DSystems), as recommended by the manufacturer and previouslydescribed [30]. Cells were incubated for 24 h with mediacontaining different concentrations of Ulva polysaccharides(0–100 μg ml−1). The supernatants were then obtained andused to determine the PGE2 levels. In some assays, indo-methacin (10 μg ml−1), a prostaglandin synthetase inhibitor,was added to the incubation medium. Standard PGE2 (dilutionseries from 1000 to 7.8 pg ml−1 in an assay buffer) was used toproduce a standard curve.

2.6. NOS-2 and COX-2 expression

RAW264.7 macrophages (107 cells in one ml) wereincubated for 24 h at 37 °C in IMDM containing 25, 50 and100 μg ml−1 of Ulva polysaccharides. Total RNAwas isolatedas described above and dissolved in DEPC-treated RNase-freewater at 1 μg ml−1. Synthesis of cDNA was accomplished aspreviously described [31]. The resulting cDNA was ampli-fied by PCR, using a specific murine NOS-2 forward/re-verse primer pair, 5′-TGGAAGCCGTAACAAAGGAAA-3′/5′-ACCACTCGTACTTGGGATGCT-3′ (NCBI accessionnumber U03699), and a specific COX-2 forward/reverseprimer pair, 5′TGATCGAAGACTACGTGCAAC-3′/5′-GCCAAAGTTGTCATGGATG-3′ (NCBI, accession numberNM_011198) [32]. Amplification of β-actin with the forward/reverse primer pair, 5′-ATGTGCAAAAAGCTGGCTTTG-3′/5′-ATTTGTGGTGGATGATGGAGG-5′ (NCBI accession n°XM_141861), was performed to control for gene expression.Thermal cycling conditions were as follows: initial denaturingat 94 °C for 5 min; then 30 cycles at 94 °C for 30 s; 55 °C for44 s; 72 °C for 1 min, and, finally a 7-min extension phase at72 °C. In all experiments, RT-PCR controls were performedwithout RNA or without RT; amplification products were notobtained in any of the experiments. The PCR products wereseparated on 2% (w/v) agarose gel stained with ethidiumbromide and photographed with a digital camera under UVtransillumination. Bands were quantified in tagged image fileformat (TIFF) with densitometry analysis software (Image-Master TotalLab, GE Healthcare, USA).

2.7. cDNA expression arrays

A mouse cDNA microarray containing up to 96 genesassociated with inflammatory cytokines and arrayed in a tetra-spot pattern, was used (cDNA GEArray™, Superarray,Bioscience Corporation). The genes in the array and theprotocols used for hybridization appear on the manufacturer'swebsite (www.superarray.com). The AmpoLabeling-LPR kit(Superarray) was used for probe synthesis. Conversion of RNAto biotinylated labelled cDNA probes was achieved by retro-transcriptase reaction (RT) and linear polymerase reaction(LPR). The labelled cDNA probe was denatured by heating at94 °C for 5 min. The arrays were prehybridized at 60 °C for 2 h

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with GEA hybrydization solution containing 100 μg/ml of heat-denatured sheared salmon-sperm DNA, then hybridized over-night in GEA hybridization solution containing the biotin-labeled cDNA probes, at 60 °C with continuous shaking. Afterhigh and low stringency washes with SSC/SDS solution at60 °C, the hybridized membranes were prepared for chemilu-minescence detection, carried out at room temperature. Themembranes were blocked for 40 min in GEA blocking solution,incubated with a 1:7500 dilution of alkaline phosphatase-conjugated with streptavidin for 10 min, washed five times withwashing buffer, then twice with AP-assay buffer, and incubatedfor 2 min with 1 ml of CDP-Start (Tropix, USA) chemilumi-nescent substrate. Finally, themembraneswere exposed toX-rayfilm, which was scanned to obtain a TIFF image file. Forquantification of gene expression, results were expressed asdigital light units (DLUs). Spots on the arrays were quantified bythe grid analysis included in the ImageMaster TotalLab softwarepackage (GE Healthcare, USA), with subtraction of backgrounddefined as the signal from a blank. To allow comparison withdata from other arrays, all signals were normalized with respectto the mean signal for the housekeeping genes included in theDNA array (PUC18 PlasmidDNA, glyceraldehyde-3-phosphatedehydrogenase, peptidylprolyl isomerase A, ribosomal proteinL13a, and cytoplasmic beta actin).

2.8. Data presentation and statistical analysis

Data are expressed as mean values±standard errors (SEM).Statistical comparisons (α=0.05) were performed by one-wayANOVA followed by the Tukey–Kramer test for multiplecomparisons. Statistical comparisons of signals obtained inmicroarrays were performed by Student's two-tailed t-test, aspreviously described [33]. In the gene arrays used in the presentstudy, those genes that were not expressed or that were expressedat very low levels in control macrophages (less than 10chemiluminiscence units), or at very low levels in stimulatedmacrophages (less than 25 chemiluminiscence units) were notconsidered in the analysis. In the remaining genes analysed, upor down-regulation was considered as significant when thelevels of expression were more than 2 higher or less than 0.5lower than in control macrophages.

3. Results

3.1. Components of the acidic sulphated polysaccharides used

Acidic polysaccharides were obtained from protein freewater-soluble extracts (PF-WSE) of U. rigida collected on thecoast of Galicia (NW Spain), as described in a previous study[24,25]. The PF-WSE were fractionated into neutral and acidsugars by anion exchange chromatography. Analysis by gelpermeation chromatography revealed that the acidic poly-saccharides mainly contained polysaccharides of high molec-ular mass (about 2000 kDa). Chemical analysis revealed thatthe sugar contents of the acidic sulphated polysaccharides usedin the present study mainly comprised rhamnose (mol.%, 48)

and uronic acids (mol.%, 46). The structures of the acidicpolysaccharides and the desulphated fractions were comparedby infrared spectroscopy. As previously described [25], thespectra obtained showed strong S=O absorption bands at 1260,850 and 792 cm−1 that disappear after desulphation ofpolysaccharides. The molar ratio of the polysaccharidecomponents was not significantly affected by the desulphationprocess (data not shown). The samples were also analysed by1H-NMR spectroscopy, which revealed that, in addition touronic acid, the U. rigida polysaccharides contained sulphatedrhamnose, xylose and sulphated xylose, as the chemical shiftswere similar to those previously found for ulvan [18]. A moredetailed characterisation of U. rigida oligosaccharides showedthat ulvan containedmainly [→4-β-D-glucuronic acid-(1→4)-α-L-rhamnose 3-sulphate-1→] and short blocks of [→4-β-D-xylose-(1→4)-α-L-rhamnose 3-sulphate-1→] and [→4-β-D-xylose 2-sulphate-(1→4)-α-L-rhamnose 3-sulphate-1→] [18].

3.2. Nitric oxide (NO) production

In previous studies, we have found that the acidic sulphatedpolysaccharides obtained fromU. rigida had potent stimulatoryeffects on phagocytes of certain fish species [24,25], animalmodels that we have used to analyse the effects of immu-nostimulants on the immune system. We believe that thepolysaccharides isolated from U. rigida may also havestimulatory effects on mammalian phagocytes, despite theevolutionary distance between fish and mammals. Becauseulvans resemble glycosaminoglycans, substances that can beproduced by activated macrophages during inflammatoryresponses, and because macrophages play a central role in theinnate and acquired immune responses, we investigatedwhether the acidic sulphated polysaccharides obtained fromU. rigida could modify the activity of RAW264.7 murinemacrophages in vitro. Firstly, we determined the effect ofU. rigida acidic polysaccharides on production of mediators ofinflammation such as nitric oxide, a molecule that participatesin many physiological events during inflammation and that canbe produced in considerable amounts by stimulated RAWmacrophages. We found that the amount of nitrites inmacrophage supernatants increased considerably in cellsincubated with polysaccharides. The response increasedprogressively with the concentration of polysaccharides andwas effectively blocked by the NO synthase inhibitor NMMA(Fig. 1). In preliminary studies, we found that the production ofNO reached a plateau at 100 μg/ml of polysaccharides and thatconcentrations lower than 25 μg/ml had very little effect onmacrophage activity. Therefore, concentrations of polysacchar-ides ranging from 25–100 μg/ml were used in all subsequentexperiments. TheU. rigida polysaccharides used in the presentexperiments are heavily sulphated and elimination of thesulphate group may affect the activity of these polysaccharides.We found that desulphation of U. rigida polysaccharidesreduced the stimulatory effects and the cells incubated withthese polysaccharides released about 50% less NO than thecells incubated with sulphated polysaccharides. NO productionwas synergistically enhanced when the RAW cells were

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stimulated by the combination of IFN-γ and LPS. Wedetermined if U. rigida polysaccharides could modulate theeffects of these substances on NO production in RAWmacrophages. Polysaccharides did not significantly enhanceNO production in the cells stimulated with the combination ofLPS plus IFN-γ, perhaps because nitrite production in cellsstimulated onlywith the latter substances was already very high(Fig. 1).

3.3. NOS-2 expression following stimulation withpolysaccharides

After confirming that RAW264.7 cells stimulated withpolysaccharides showed increased NO production, we tried todetermine if the enhanced NO production was attributable toan increase in the expression of the inducible isoform of nitricoxide synthase (NOS-2), an enzyme that is expressed at high

levels in activated murine macrophages. We also found thatRAW264.7 murine macrophages incubated with several acidicsulphated polysaccharides for 24 h produced more NOS-2mRNA than control cells, in a dose-dependent manner (Fig. 2).

3.4. PGE2 secretion

Prostaglandins are important mediators of the inflammatoryresponses, and modulate the production of inflammatorycytokines; macrophages are one of the major sources of PGE2.We analyzed the effect of polysaccharides on the ability ofRAW264.7 macrophages to secrete PGE2. Release of PGE2 intothe supernatant by both control and stimulated macrophagesincreased significantly, in a dose-dependent manner, when themacrophages were incubated with polysaccharides for 24 h.Secretion was inhibited when indomethacin, a prostaglandinsynthetase inhibitor, was added to the incubationmedium (Fig. 3).

3.5. COX-2 expression following stimulation withpolysaccharides

Synthesis of prostaglandins is dependent on the activity ofcyclooxygenase, and COX-2 (an inducible form of cycloox-ygenase) is thought to play an important role in inflammation, asmacrophages expressCOX-2 in response to a variety of cytokines

Fig. 2. Levels of mRNA in NOS-2 and COX-2 in RAW264.7 murinemacrophages incubated withU. rigida polysaccharides (0–100 μg ml−1)for 24 h. A) Agarose gel (2%) analysis of RT-PCR products with primersfor NOS-2, COX-2 and the housekeeping gene β-actin (330 bp).B) Levels of mRNA as determined by densitometric analysis [opticaldensity (OD) of bands of NOS-2 and COX-2 divided by OD of bandobtained in parallel assay using primer for β-actin]. Values shown aremeans±SD (n=3 assays). ⁎Pb0.05 relative to the control (0 μg ml−1).

Fig. 1. Nitric oxide production by stimulated RAW264.7 murinemacrophages. The macrophages were stimulated for 48 h with sulphatedor desulphated Ulva rigida polysaccharides (0–100 μg ml−1) in culturemedium, in the absence (A) or presence (B) of IFN-γ (10 U/ml) and LPS(LPS) (100 ng ml−1). The arginine analogue N(G)-monomethyl-L-arginine (L-NMMA) (250 μM)was also added to the incubation mediumin some flasks. Each point is the mean±SDof quintuplicate experiments.⁎Pb0.01 relative to the control; ⁎⁎Pb0.01 relative to desulphatedpolysaccharide.

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as well as LPS. PGE2 synthesis is regulated by COX-2 and weinvestigated whether COX-2 expression varied in macrophagesstimulated with polysaccharides. We found that acidic sulphatedpolysaccharides were very potent inducers of COX-2 geneexpression in RAW264.7 murine macrophages, stimulation ofwhich produced very high levels of COX-2 mRNA, in a dose-dependent manner (Fig. 2).

3.6. Effects of acidic sulphated polysaccharides on macro-phage gene expression

Having shown that U. rigida acidic polysaccharides inducean increase in the expression of NOS-2 and COX-2 in RAWcells, we wondered if these polysaccharides would also affectthe expression of other genes involved in inflammatoryresponses. We used a commercial cDNA microarray contain-ing 96 genes encoding for mouse inflammatory cytokines andreceptors to study the gene expression in response toincubation with the acidic polysaccharides. Total RNA wasisolated from non-stimulated or stimulated macrophages.Macrophages were incubated for 24 h with 50 μg ml−1 ofpolysaccharides, a concentration that induced good expressionof NOS-2 and COX-2. The gene expression patterns found incontrol and in RAW246.7 macrophages incubated with Ulvapolysaccharides are shown in Table 1. Data were distributedinto four groups in relation to the gene expression in controlmacrophages. Of those genes expressed at low levels in controlmacrophages (10 to 50 fluorescence units), 5 were up-regulated by more than two times in stimulated macrophages,and included chemokine (C motif) ligand 1 (Xcl1), chemokine(C-X-C motif) ligand 12 (Cxcl12), chemokine (C-C motif)ligand 22 (Ccl22) and IL6 signal transducer (IL6st). In genesexpressed at intermediate or high levels in control macro-phages, IL12 receptor beta 1 (IL12rb1) and chemokine (C-X-Cmotif) ligand 14 (Cxcl14) were also up-regulated by more than

Fig. 3. Levels of PGE2 in medium after incubation of RAW264.7murine macrophages with U. rigida sulphated polysaccharides for24 h, in the absence or presence of indomethacin (10 μg ml−1). Eachpoint is the mean±SD of quintuplicate experiments. ⁎Pb0.01 relativeto the control.

Table 1Microarray analysis of genes up or down-regulated in RAW264.7macrophages following stimulation with Ulva rigida sulphatedpolysaccharides

Mean fold change

Genes not expressed or with very low expression (0–10 DLUs) incontrol macrophages

Chemokine (C motif) ligand 1 (Xcl1)Chemokine (C-C motif) ligand 22 (Ccl22)Chemokine (C-C motif) ligand 8 (Ccl8)Chemokine (C-C motif) receptor 6 (Ccr6)Chemokine (C-X-C motif) ligand 12 (Cxcl12)Chemokine (C-X-C motif) ligand 15 (Cxcl15)Expressed sequence BF534335Interleukin 5 receptor, alpha (Il5ra)Interleukin 6 signal transducer (Il6st)Interleukin 12A (Il12a)Interleukin 12B (Il12b)Interleukin 13 receptor, alpha 2 (Il13ra2)Interleukin 15 (Il15)Interleukin 15 receptor, alpha (Il15ra)Interleukin 17 receptor (Il17r)Interleukin 17B (Il17b)Lymphotoxin A (Lta)Macrophage migration inhibitory factor (Mif)Transforming growth factor, beta 2 (Tgfb2)Transforming growth factor, beta 3 (Tgfb3)Tumor necrosis factor receptor superfamily, member 1a(Tnfrsf1a)

Genes with low expression (10–50 DLUs) in control macrophagesChemokine (C-C motif) ligand 3 (Ccl3) 3Stromal cell derived factor 2 (Sdf2) 2.7Tumor necrosis factor receptor superfamily, member 1b(Tnfrsf1b)

2.3

Chemokine (C-X-C motif) ligand 10 (Cxcl10) 2.2Interleukin 4 (Il4) 2.1Chemokine (C-C motif) ligand 9 (Ccl9) 2Interleukin 5 (Il5) 2Chemokine (C-X-C motif) ligand 9 (cxcl9) 1.9Chemokine (C-C motif) ligand 6 (Ccl6) 1.9Interleukin 13 receptor, alpha 1 (Il13ra1) 1.8Chemokine (C-C motif) receptor 1 (Ccr1) 1.7Interleukin 10 (Il10) 1.7Interleukin 20 (Il20) 1.7Chemokine (C-X-C motif) ligand 11 (Cxcl11) 1.7Interleukin 2 receptor, alpha chain (Il2ra) 1.7Transforming growth factor, beta 1 (Tgfb1) 1.6Interleukin 10 receptor, alpha (Il10ra) 1.6Chemokine (C-C motif) receptor 4 (Ccr4) 1.6Chemokine (C-C motif) ligand 2 (Ccl2) 1.6Interleukin 11 receptor, alpha 1 (Il11ra1) 1.6Small chemokine (C-C motif) ligand 11 (Ccl11) 1.5Interleukin 1 beta (Il1b) 1.5Interleukin 9 receptor (IL9r) 1.5Interleukin 18 receptor 1 (Il18r1) 1.5Chemokine (C-X3-C motif) ligand 1 (Cx3cl1) 1.5Interleukin 9 (Il9) 1.2Interleukin 2 receptor, gamma chain (Il2rg) 1.2Chemokine (C-C motif) ligand 21c (leucine) (Ccl21c) 1Chemokine (C-C motif) ligand 7 (Ccl7) 0.7

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two times in stimulated macrophages. In stimulated macro-phages no genes were expressed at levels of less than 0.5 timesthe response of control macrophages. These data suggest that

U. rigida polysaccharides induce stimulation of RAWmacrophages, thereby increasing the expression of severalgenes related to inflammatory responses.

4. Discussion

The effects of U. rigida acidic sulphated polysacchar-ides on the biological activities of RAW264.7 murinemacrophages were evaluated in the present study. Aspreviously shown [24,25], the results of chemicalanalyses and of the infrared spectral and 1H-NMRspectroscopic analyses indicate that the structure of theacidic sulphated polysaccharides used in the present studyis similar to that of ulvan [34]. Ulvan obtained fromU. rigida contains repeated disaccharide units describedas β-D-glucuronosyluronic acid (1→4) L-rhamnose 3-sulphate [18,19]. The particular structure of the ulvan andits resemblance to glycosaminoglycans make it worth-while to investigate whether this molecule has anybiological activity in mammals. The results of the presentstudy show that U. rigida polysaccharides stimulatedRAW264.7 macrophages by modifying several activitiesin these cells, e.g. they caused an increase in the release ofPGs or NO and the up-regulation of the expression ofseveral genes associated with pro-inflammatory reactionssuch as those involving chemokines.

U. rigida polysaccharides induced an increase in theproduction of NO by macrophages and in the expressionof NOS-2, an enzyme that metabolises L-arginine toL-citrulline and NO. The biological activities of NOarewidespread and this compound plays a complex role inthe regulation of the inflammatory response and exertseffects on the function of several cell types including theantimicrobial activity of phagocytes [35–37]. Macro-phages incubated with U. rigida polysaccharides alsocontained higher levels of PGE2 than controls and showedincreased expression of COX-2, an inducible enzyme thatcatalyses the conversion of arachidonic acid to prosta-glandins. These results suggest that the increased PGE2production is mainly due to over-expression of COX-2.Prostaglandin E2 is produced by many cell types,including macrophages, and participates in the regulationof immune and inflammatory responses that havecomplex effects on cells such as macrophages, lympho-cytes or NK cells [38]. It is produced at the initial phase ofthe inflammatory response, thereby inducing vasodilata-tion and increased blood vessel permeability [39,40].

After incubation of macrophages with polysacchar-ides, there was a more than 2-fold increase in the ex-pression of three chemokine ligands (Ccl3, Cxcl10 andCxcl14). These chemokines play an important role ininflammatory responses by inducing the recruitment of

Table 1 (continued)

Mean fold change

Genes with low expression (10–50 DLUs) in control macrophagesChemokine (C-C motif) receptor 3 (Ccr3) 0.6

Genes with medium expression (50–100 DLUs) in control macrophagesInterleukin 12 receptor, beta 1 (Il12rb1) 2.4Chemokine (C-C motif) receptor 7 (Ccr7) 1.9Interleukin 17 (Il17) 1.7Chemokine (C-C motif) ligand 25 (Ccl25) 1.7Chemokine (C-C motif) ligand 17 (Ccl17) 1.5Chemokine (C-C motif) ligand 4 (Ccl4) 1.4Chemokine (C-X-C motif) ligand 5 (Cxcl5) 1.4Interleukin 6 (Il6) 1.4Interleukin 11 (Il11) 1.4Interleukin 1 alpha (Il1a) 1.4Interleukin 12 receptor, beta 2 (Il12rb2) 1.3Interleukin 2 (Il2) 1.3Chemokine (C-X3-C) receptor 1 (Cx3cr1) 1.2Chemokine (C-motif) receptor 1 (Xcr1) 0.9Chemokine (C-C motif) receptor 2 (Ccr2) 0.8Burkitt lymphoma receptor 1 (Blr1) 0.7Chemokine (C-C motif) receptor 8 (Ccr8) 0.7Chemokine (C-X-C motif) receptor 3 (Cxcr3) 0.6

Genes with high expression ( N100 DLUs) in control macrophagesChemokine (C-X-C motif) ligand 14 (Cxcl14) 2.1Interleukin 8 receptor, beta (IL-8Rb), 2Chemokine (C-X-C motif) ligand 13 (Cxcl13) 1.7G protein-coupled receptor 2 (Gpr2) 1.5Chemokine (C-X-C motif) ligand 1 (Cxcl1) 1.5Interleukin 16 (Il16) 1.4Interleukin 10 receptor, beta (Il10rb) 1.3Chemokine (C-C motif) ligand 20 (Ccl20) 1.3Interleukin 18 (Il18) 1.2Interleukin 1 receptor, type I (Il1r1) 1.2Interleukin 2 receptor, beta chain (Il2rb) 1.2Interferon gamma (IFNg ) 1.2Lymphotoxin B receptor (Ltbr) 1.2Interleukin 1 receptor, type II (Il1r2) 1.1Interleukin 13 (Il13) 1.1Chemokine (C-X-C motif) ligand 2 (Cxcl2) 1.1Tumor necrosis factor (Tnf) 1.1Chemokine (C-C motif) ligand 5 (Ccl5) 1.1Lymphotoxin B (Ltb) 1.1Interleukin 21 (Il21) 1Chemokine (C-C motif) ligand 24 (Ccl24) 1Chemokine (C-C motif) ligand 19 (Ccl19) 1Chemokine (C-C motif) receptor 5 (Ccr5) 0.9Interleukin 6 receptor, alpha (Il6ra) 0.9Chemokine (C-C motif) ligand 12 (Ccl12) 0.9Transforming growth factor, alpha (Tgfa) 0.9Chemokine (C-X-C motif) receptor 4 (Cxcr4) 0.7

Mean fold change = mean stimulated/control macrophages. The MeanFold Change in those genes that were not expressed or had very lowlevels of expression in control and stimulated macrophages is not shown.

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leucocytes to sites of injury. Ccl3 is a β-chemokine thatregulates the migration of leucocytes such as monocytes,T cells, neutrophils, eosinophils, basophils, and NK cells[41] and increases the capacity of macrophages to killseveral pathogens [42,43]. Cxcl10 is a proinflammatorychemokine that is mainly produced by macrophagesstimulated with interferon gamma (IFN-γ) and that ischemotactic for monocytes and for T cells [44]. Cxcl14binds specifically to B-cells and macrophages andsubcutaneous injection of the chemokine generates aninflammatory response in Nude and C3H/HeJ mice [45].

An increase in the expression of interleukin 12 receptorbeta 1 and TNF receptorwas also observed. Interleukin 12receptor beta 1 is expressed in cells of haematopoieticorigin, including NK cells, activated T-cells, dendriticcells and microglia [46,47]. This receptor recognizes IL-12, a family of cytokines with important pro-inflamma-tory and immunoregulatory functions. The TNFRSF1Bgene encodes a tumour necrosis factor receptor, which ishighly expressed in osteoclast precursors and plays animportant role in mediating the effects of TNF inosteoclastogenesis [48].

The observed effects of ulvan on macrophage geneexpression and on the production of molecules such usPGE2 and NO appear to indicate that ulvan modulates theactivity of RAW264.7 macrophages in vitro by inducingthe release of inflammatory molecules. As mentionedabove, ulvan from U. rigida is made up of disaccharide-repeating units composed mainly of glucuronic acid andrhamnose, and about 95% of the rhamnose is sulphated[34]. Interestingly, we found that the stimulatory capacitywas lost when polysaccharides were desulphated. Similarresults were obtained when the effects of ulvan were testedon fish phagocytes [25]. It was also reported that othersulphated polysaccharides such us porphyran, obtainedfrom other seaweed species, also stimulated mice macro-phages, but that this activity decreased considerably afterdesulphation, suggesting that the sulphate groups arenecessary for macrophage stimulation through directinteraction between these groups and cells, or indirectlyby maintaining the conformation of the active moieties[8,9]. The polysaccharides used in the present study areprobably too large to cross the cell membrane ofRAW264.7 macrophages, and stimulation may occurthrough interaction between the molecules and surfacereceptors, with the sulphate groups playing a role in thisinteraction; these aspects require further investigation.Other sulphated polysaccharides obtained from seaweedalso stimulate macrophages. Carrageenan, a sulphatedpolygalactose obtained from red seaweed, is considered asan inflammatory agent [4]. There are several types ofcarrageenan that differ in total sulphate-content distribution

and molecular configuration. As with ulvan in the presentstudy, some carrageenans induce the synthesis of pros-taglandins and NO by macrophages [5,49,50]. Activationof macrophages by lambda-carrageenan is dependent onToll-like receptor-4 [12]. Ulvan probably induces macro-phage stimulation in a similar way to some of thecarrageenans, although the involvement of receptors,such us Toll-like receptors, in the process of macrophageactivation by ulvan has yet to be demonstrated.

As mentioned above, ulvan shares certain structuralsimilarities with glycosaminoglycans (GAGs). The latterare released at inflammatory sites by activated macro-phages and after degradation of extracellular matrix, andGAGs such as chondroitin sulphate are produced byinflammatory macrophages [21] and by polymorphonu-clear leucocytes after phagocytosis [51]. Chondroitinsulphates have been reported to be inflammatory me-diators that can activatemonocytes, in a response thatmaybe mediated by the CD44 receptor [22]. Hyaluronan andchondroitin sulphate oligosaccharides modulate IL-12production in macrophages, although the effects dependon how the oligosaccharides are obtained [52]. Althoughin the present study no increase in the production of thepro-inflammatory cytokine IL-12 was observed, theresults also suggest that ulvan induced an inflammatoryresponse in macrophages. As mentioned above for car-rageenans, it is not known whether the GAGs and ulvaninteract with macrophages by using the same receptor;this is another interesting aspect that must be explored infuture research.

In summary, the results obtained in the present studyindicate that ulvan can modulate the activity ofRAW264.7 murine macrophages and that the presenceof sulphate groups in the molecule appears to benecessary. U. rigida acidic sulphated polysaccharidesmay be used as inflammatory agents because of theirstructural similarity to carrageenans, although they mayproduce more complex activities because of theirsimilarity to some GAGs. The present data also suggestthat these polysaccharides are of potential interest asstimulators of macrophages in diseases where macro-phage function is impaired or needs to be enhanced.

Acknowledgements

The present study was financially supported by theMinisterio de Educación y Ciencia, Spain (AGL2004-01896).We thank Professor Ignacio Zarra (Departamentode Biología Vegetal, Universidad de Santiago deCompostela) for helping us to characterize the polysac-charides, and Dr. José Antonio Abelenda for carrying outthe 1H-NMR analysis of the samples.

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