55 Modulation by extracts of the dicentrarchi of turbot leucocyte functions and inflammatory cytokine gene expression.

Leiro, J.; Paramá, A.; Arranz, J.A.; Álvarez, M.F. and Sanmartín M.L. Instituto de Investigación y Análisis Alimentarios, Laboratorio de Parasitología, Universidad de Santiago de Compostela, 15782, Santiago de Compostela, Spain.

Received: 24.11.05 Accepted: 12.12.05

Abstract: Philasterides dicentrarchi is a causing in farmed turbot. This study shows that excretion‑secretion products (ESP) and a total cilitate lysate (TCL) of this parasite have different effects on the responses of thioglycollate‑induced turbot peritoneal leucocytes. Specifically, leucocytes incubated in vitro with ESP showed increased chemotaxis and increased NO production, while leucocytes incubated in vitro with TCL showed increased phagocytic activity, increased production of reactive oxygen species (ROS), but reduced NO production. Intracellular levels of mRNAs of the pro-inflammatory cytokines interleukin 1 beta (IL‑1β) and tumour necrosis factor alpha (TNF‑α) peaked about 3 days after inoculation with thioglycollate, while mRNA levels of the anti‑inflammatory cytokine transforming growth factor beta (TGF‑β) peaked within the first 24 hours. Inoculation of turbot with 3% thioglycollate plus ESP led to increased intraleucocyte mRNA levels of IL‑1β, while inoculation with 3% thioglycollate plus TCL led to increased mRNA levels of TGF‑β. These results indicate that live and dead Philasterides dicentrarchi have various effects on leucocyte function and on the expression of immunoregulatory genes. It seems likely that these immunomodulatory effects will have important consequences for the inflammatory response generated during natural infection of turbot with this parasite. Keywords: turbot, Philasterides dicentrarchi, ciliate, leucocytes, respiratory burst, proinflammatory cytokines

Resumen: Philasterides dicentrarchi es un ciliado que provoca la scuticociliatosis en el rodaballo en cultivo. Este estudio demuestra que los productos de excrección-secrección (ESP) y un lisado total del ciliado (TCL) de este parásito produce diferentes efectos sobre la respuesta de leucocitos peritoneales de rodaballos inducidos con tioglicolato. Específicamente, los leucocitos incubados in vitro con ESP mostraron un incremento en la quimiotaxis y en la producción de óxido nítrico (NO), mientras que los leucocitos incubados in vitro con TCL presentaron un incremento en la actividad fagocítica y en la producción de especies reactivas del oxígeno (ROS), pero una reducción en la producción de NO. Los niveles intracelulares de ARNm de las citocinas proinflamatorias interleucina 1 beta (IL-1b) y el factor de necrosis tumoral alfa (TNF-a) presentaron un pico de expresión al tercer día tras la inoculación con tioglicolato, mientras que los niveles de ARNm de la citocina factor transformante del crecimiento beta (TGF-b) alcanzó la máxima producción alrededor de las primeras 24 horas. La inoculación de rodaballos con tioglicolato junto con ESP incrementó los niveles de ARNm de IL-1b, mientras que la inoculación con tioglicolato y TCL incrementó los niveles de TGF-b. Estos resultados indican que según el estado de viabilidad P. dicentrarchi puede inducir diferentes efectos sobre la función leucocitaria y sobre la expresión de los genes inmunoreguladores. Parece probable que estos efectos inmunomoduladores produzcan importantes consecuencias sobre la respuesta inflamatoria generada durante la infección natural del rodaballo por este parásito. Palabras clave: rodaballo, Philasterides dicentrarchi, ciliado, leucocitos, estrés oxidativo, citocinas proinflamatorias.

1. Introduction

Corresponding author: The euryhaline histiophagous ciliate Philasterides Dr. José Manuel Leiro Vidal dicentrarchi was initially described as an opportunistic Laboratorio de Parasitología, parasite causing systemic infections in Dicentrarchus Instituto de Investigación y Análisis Alimentarios (I.I.A.A.), labrax in Mediterranean coastal lagoons (Dragesco et al., Universidad de Santiago de Compostela, 1995), but recently it has been recognized as an emergent C/ Constantino Candeira s/n, 15782, Santiago de Compostela, Spain. infectious agent causing important economic losses in Tel. +34-981-563100 farmed turbot, Scophthalmus maximus, on the Atlantic Fax: +34-981-547171 e-mail: [email protected]

Revista Ibérica de Parasitología (2006), 66 (1-4), 55-67. © 2006 Sociedad Española de Parasitología (SEP) 56 Leiro, J. et al., Leucocyte functions and cytokine responses in turbot scuticociliatosis coast of northwest Spain (Iglesias et al., 2001; Iglesias et fish were acclimatized for at least 15 days in 10-l tanks al., 2002). P. dicentrarchi causes severe systemic disease with a constant flow of water (18 ± 1ºC, pH 6.5 ± 0.5) in turbot, acting as an endoparasite that divides rapidly in and aeration. The fish received a standard semi-dried many organs and feeds on cells (mainly erythrocytes) and pelleted food daily. Blood samples were collected by other tissue components, causing the death of the host caudal vein puncture from fish anaesthetized with 0.03% (Iglesias et al., 2003 b). The presence of in internal 2-phenoxy ethanol (Sigma). The serum was separated organs is associated with intense oedematous inflammation by centrifugation at 2000 x g for 10 min, and stored at due to accumulation of inflammatory cellular infiltrate -30ºC until use. (principally monocytes and lymphocytes), especially in the areas showing the highest densities of P. dicentrarchi 2.2. Parasites and culture trophozoites (Iglesias et al., 2001). The ciliate Philasterides dicentrarchi (Iglesias Phagocytosis is the primordial defense mechanism et al., 2001) was obtained from ascitic fluid of naturally of all metazoan organisms, including , to infected turbot, and maintained axenically at 18ºC in infectious organisms such as parasites (Neuman et ‘complete’ L-15 medium (Leibovitz, 10‰, salinity, pH al., 2001). Phagocytic cells act in phases: chemotaxis, 7.2; Iglesias et al., 2003a), containing 90 mg/l each of attachment to the parasite, ingestion, killing and digestion adenosine, cytidine and uridine, 150 mg/l of guanosine, 5 (Verhoef, 1991). Killing of parasites by phagocytes may g/l of glucose, 400 mg/l of L-a-phosphatidylcholine, 200 be oxygen-dependent (respiratory burst) or oxygen- mg/l of Tween 80, 10% heat-inactivated foetal bovine independent (Verhoef, 1991). The respiratory burst, serum (FBS) and 10 ml/l of 100 X antibiotic antimycotic induced by chemotactic stimulation or phagocytosis, solution (= 100 units/ml of penicillin G, 0.1 mg/ml of involves release of reactive oxygen intermediates (ROS) streptomycin sulphate and 0.25 mg/ml of amphotericin that kill parasites, but that may also cause tissue damage B) (all from Sigma-Aldrich, USA). and inflammation (Bagglioni & Wymann, 1990). ROS are potent inducers of the pro-inflammatory stress response 2.3. Stimuli, drugs and chemicals typified by pro-inflammatory cytokines, prostaglandins, Total ciliate lysate (TCL) was obtained as thromboxanes, leukotrienes, leukocyte adhesion previously described (Iglesias et al., 2003b). Briefly, molecules and chemokine synthesis (Tse et al., 2004). ciliates collected by centrifugation at 650 x g for 5 min Although very limited information is available on the were resuspended in saline phosphate buffer (PBS; effect of scuticociliate infections on the fish non-specific 0.015 M phosphate buffer, 0.15 M NaCl, pH 7.2) immune system, recently we have demonstrated that containing 0.1 mM pepstatin A, 0.02 mM N-(trans- infection of turbot with the ciliate P. dicentrarchi has the epoxysuccinyl)-L-leucine 4 guanidinobutylamide (E-64), following effects on phagocytic cells: (a) modulation of 1 mM phenylmethanesulfonyl fluoride (PMSF) and 2 complement activation and intracellular ROS production, mM ethylenediaminetetraacetic acid (EDTA) as protease (b) slightly increased NO production, and (c) increased inhibitors (all from Sigma-Aldrich). Subsequently, scavenging of extracellular ROS (Leiro et al., 2004). the ciliates contained in the suspension were lysed At present, there is no precise information of the ultrasonically in a Branson W-250 sonifier (Branson modulatory effects of P. dicentrarchi extracts on phagocyte Ultrasonic Corporation, USA). The whole process was function or inflammatory responses in turbot. The aim performed on ice. The resulting lysate (TCL) was stored of the present study was to investigate the modulatory at -80 ºC. effects of two P. dicentrarchi components, a total ciliate Excretion-secretion products (ESP) were lysate (TCL) and excretion-secretion products (ESP), obtained as previously described (Paramá et al., 2004). on the main phagocytic functions (namely chemotaxis, Briefly, 2 x 6 10 cells/ml where incubated for 24 h at phagocytosis, and the respiratory burst) and the expression 18ºC in ‘incomplete’ L-15 medium (like ‘complete’ of genes related to the main cytokines involved in the L-15 medium but without nucleosides, glucose, lipids inflammatory process (interleukin 1 beta, IL-1b; tumour and FBS). The suspension was filtered through 0.22m m necrosis factor alpha, TNF- ; and transforming growth a sterile filters (Iwaki, Japan) and the filtrate concentrated factor beta, TGF- ). b 25 times using YM-10 Microcon centrifugal filter devices (Millipore, USA). The resulting concentrate (ESP) was 2. Materials and Methods stored at -80ºC until use. Protein concentration in TCL 2.1. Fish and ESP was determined by Bradford’s method (1976) Juvenile turbot Scophthalmus maximus L. (50- using a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, 100 g) were obtained from a local farm in the north of Germany), with bovine serum albumin (Sigma-Aldrich) Galicia (north-western Spain). Prior to experiments, the as standard. Leiro, J. et al., Leucocyte functions and cytokine responses in turbot scuticociliatosis 57

Blood samples were collected by caudal vein puncture (Sterilin, England), using a capillary tube with inverted from fish anaesthetized with 0.03% 2-phenoxy ethanol U shape to connect two wells (A and B) containing A) (Sigma), and the serum was separated by centrifugation at 200 ml of peritoneal leucocytes (3 x 106 cell/ml) in L-15 2000 x g for 10 min, and stored at -30ºC until use. medium and B) 200 µl of a solution of chemoattractant in Kluyveromyces lactis (strain NRRL-41140) cells L-15 medium. Migration of cells to the chemoattractant were cultured on 1% yeast extract, 2% peptone, 2% was assessed after 4 h incubation at 23ºC: leucocytes glucose (YPD medium; Difco, USA) at 30ºC, at a growth present inside the tubes and in the B wells were fixed rate of 0.07/h, then washed with distilled water and with 1% glutaraldehyde, and the number of cells was finally freeze-dried. Stock solution (2 mg/ml) of phorbol determined using a Neubauer haemacytometer. Migration 12-myristate 13-acetate (PMA) (Sigma, St Louis, MO, was expressed as chemotaxis index, i.e. number of USA) was dissolved in dimethylsulfoxide (DMSO) and migrated cells/random migration (random migration is stored in the dark at -80ºC until use. Stock solution of defined as the number of cells that migrated towards the lipopolysaccharide (LPS) from Escherichia coli serotype assay medium without chemoattractant in parallel control 0111:B4 (Sigma) was made up at 10 mg/ml in phenol- assays). All assays were performed in quintuplicate. red-free Dulbecco’s Eagle medium (DMEM; Sigma) and stored in the dark at -20ºC until use. Thioglycollate broth 2.6. Peroxidase activity (Merck, Germany) was prepared to a concentration of 3% Leucocytes present inside chemotaxis assay tubes (w/v) in L-15 medium, autoclaved at 121ºC for 10 min, were fixed for 1 min in PBS containing 1% formaldehyde and stored at room temperature until use. The arginine and 2% glutaraldehyde, and peroxidase activity was analogue N-monomethyl-L-arginine monoacetate determined as previously described (Leiro et al., 2000a). (L-NMMA; Calbiochem, USA) was made up at 100 Briefly, fixed cells were incubated for 15 min in 0.15 M Tris mM in DMEM, and stored until use at -20ºC in the dark. buffer pH 7.6 containing 0.02% 3,3’-diaminobenzidine L-Glutamine, sulfanilamide and naphthylenediamine tetrahydrochloride and 0.006% H2O2. The slides were hydrochloride were likewise purchased from Sigma. then washed three times with Tris buffer and contrasted with haematoxylin for 4 min. After dehydration with 2.4. Elicitation and isolation of inflammatory peritoneal ethanol, permanent mounts were prepared with Eukitt. leucocytes Stained cells (dark brown cytoplasm) were counted as Inflammatory cells were elicited by i.p. injection peroxidase-positive. of fish with 1 ml of 3% Brewer thioglycollate medium in incomplete L-15 medium, in some cases also 2.7. Phagocytosis assay containing 500 mg of protein per ml of TCL or ESP. Phagocytic activity was assayed by a previously Three days later, the fish were anaesthetized as indicated described fluorometric method (Leiroet al., 2000a) using above and the anaesthesia was prolonged until dead. K. lactis yeast coupled to fluorescein isothiocyanate (FITC; Peritoneal leucocytes were then collected as described Sigma). To couple FITC, yeast cells were suspended at previously (Leiro et al., 2001), and the peritoneal fluid 109 cells ml-1 in 50 mM sodium carbonate-bicarbonate was maintained on ice. Cell concentration was counted buffer (pH 9.5) containing 150 mM NaCl and 40 mg of using a haemacytometer and adjusted in Hanks’ balanced FITC. After incubation for 1 h at room temperature, the salts solution (HBSS) to106 cells/ml by centrifugation yeast suspension was centrifuged at 10000 x g for 5 min at 500 x g for 15 min at 4ºC. Aliquots (100 ml) of the several times, each time discarding the supernatant and cell suspension were added to the wells of 96-well resuspending the pellet in PBS, until fluorescence in the microculture plates (Corning, USA) and left for 60 min supernatant dropped to zero. For assay, 100 ml of HBSS at 23ºC to allow adhesion. Non-adherent cells were then containing 106 FITC-labelled yeast cells was added to removed by two washes with 200 ml per well of HBSS, each well of 96-well microculture plates to which turbot and more than 97% of the remaining adherent cells were peritoneal leucocytes (105 per well) had been coupled as viable (as determined using the trypan blue exclusion described above, and incubated under optimal conditions test); most of these cells are neutrophils and macrophages for phagocytosis (optimal yeast-cell-to-phagocyte ratio, (Leiro et al., 2001). temperature, incubation time; Leiro et al., 1995). The wells were then washed several times with HBSS, and the 2.5. Chemotaxis assay cells were solubilized by adding 100 ml of 25 mM Tris- Chemotaxis of turbot phagocytic cells was studied HCl pH 8.5 containing 0.2% sodium dodecyl sulphate using a capillary-type microplate multiassay performed (SDS). Fluorescence was measured in a microplate as previously described (Paramá et al., 2004). The assay fluorescence reader (Bio-Tek Instruments; excitation 490 was performed in sterile 96-well flat-bottomed plates nm, emission 525 nm). 58 Leiro, J. et al., Leucocyte functions and cytokine responses in turbot scuticociliatosis

2.8. Assays of ROS production for 5 min, then centrifuged at 12000 x g for 15 min at 4ºC. Extracellular ROS release by turbot peritoneal The colourless aqueous upper phase was removed and leucocytes was quantified using the fluorogenic reagent RNA precipitated with isopropanol; the resulting RNA R pellet was dried and dissolved in diethylpyrocarbonate OxyBURST Green H2HFF BSA (Molecular Probes, The Netherlands) as previously described (Leiro et al., (DEPC)-treated RNase-free water at a concentration of 2001). Peritoneal leucocytes (100 µl, 106 cells/ml) were 1 mg/ml. cDNA synthesis (25 ml/reaction) was achieved R using 1.25 mM random hexamer primers (Roche), 250 incubated with 10 µg/ml of OxyBURST Green H2HFF BSA for 2 min at 23ºC, then stimulated by addition of 10 mM of each deoxyribonucleotide triphosphate (dNTPs), mg/ml of PMA or, in some assays, 500 mg/ml of excretion- 10 mM DTT, 20 U of RNase inhibitor, 2.5 mM MgCl2, secretion products (ESP). Fluorescence emission was 200 U of MMLV (murine leukaemia virus) reverse then determined in a microplate fluorescence reader (Bio- transcriptase (Promega) in 30 mM Tris and 20 mM KCl, Tek Instruments; excitation 488 nm, detection 530 nm, 55 pH 8.3, and 2 mg of sample RNA. The cycling parameters min). The results are expressed as increase in fluorescence for the RT step were: hybridization for 10 min at 25ºC and (arbitrary units) per min. reverse transcription for 60 min at 42ºC.

2.9. Assay of nitric oxides production 2.11. PCR amplifications, cloning and sequencing of One-hundred microliter aliquots of peritoneal turbot TGF-b leucocytes that had been prestimulated in vivo by injection Initially, TGF-b1 of turbot was amplified by PCR of 3% Brewer thioglycollate medium in L-15 medium or using an arbitrary primers set designed for amplification containing 500 mg/ml of TCL or ESP, were incubated in of murine inflammatory cytokines (Rat Set 1, Biosource Europe, Belgium) as previously described (Leiro et al., microplates (Corning) at 23ºC under 5% CO2 for 48 h in phenol-red-free DMEM containing 2 mM L-glutamine 2003). The PCR products obtained were purified and and 100 ng/ml of LPS or 500 mg/ml of ESP. In some wells, cloned in the pGEM®-T easy system (Promega, USA), the arginine analogue L-NMMA (250 mM) was included transformed in JM199 cells and sequenced as previously in the medium as a reference inhibitor of nitric oxides described (Leiro et al., 2002). Nucleotide and protein (NO•) production. sequences are deposited in GenBank (National Center for NO• production in the culture supernatants was Biotechnology Information, Bethesda, Maryland, USA) assayed by the Griess reaction (Green et al., 1982), by with accession number AF521669). measurement of the total amount of inorganic NO• as previously described (Leiro et al., 2004). Aliquots (100- 2.12. Expression analysis of pro-inflammatory cytokines ml) were removed from the medium and incubated with in turbot inflammatory peritoneal leucocytes an equal volume of Griess reagent (1% sulfanilamide and For determination of cytokine gene expression we

0.1% naphthylenediamine hydrochloride in 2.5% H3PO4) used a semi-quantitative RT-PCR (reserve transcription for 10 min at room temperature, and absorbance was then polymerase chain reaction). One ml of RT reaction mixture measured at 530 nm in an ELISA reader (Titertek Multiscan, was amplified by PCR using the following forward/reverse Flow Laboratories, Finland). Nitrite concentration was primer pair: 5’-AAGCGGCCTCTACTCCGTCTA-3’ calculated with reference to a standard curve obtained / 5’-CCCAGGTAGATGGCATTGTA-3’ (TNF-a; using NaNO2 (1-200 mM in culture medium). GenBank of the National Center for Biotechnology Information, NCBI, accession number AB040449) (Hirono 2.10. RNA isolation and cDNA production et al., 2000); 5’-ACAATTCCTGGCGTTACCTT-3’ / 5’- One-thousand-microliter aliquots of 106 peritoneal TCGGTTCATGTCATGGATGGT-5’ (TGF1-b; NCBI, accession inflammatory cells stimulated in vivo with thioglycollate number AF521669); 5’-TACCTGTCTTGCAACAGGAA-3’ / or with TCL or ESP, were used for isolation of RNA. 5’-TGATGAACCAGTTGGGGAA-3’ (IL-1b; NCBI, accession Isolation of total RNA from leucocyte samples was done number AJ295836) and 5’-AACTGGGATGACATGGAGA-3’ with a monophasic solution of phenol and guanidine / 5’-CTCAGGATCTTCATGAGGTA (b-actin, NCBI, accession thiocyanate (TriPure Isolation Reagent, Roche). Briefly, number AY0083055’-ACCGCATCTTCTTGTGCAGT-3’ / 1 ml of TriPure was added to each well (area 10 cm2), and 5’- GCCAAAGTTGTCATGGATGA-3’), to confirm gene the cells were lysed by passing the suspension through expression. These primers amplified fragments of 100 a pipette several times. The lysate was incubated for 5 bp (TNF-a), 181 bp (IL-1b), 243 bp (TGF-b) and 330 min at room temperature to ensure complete dissociation bp (b-actin). The 25-ml optimized reaction mixture of nucleoprotein complexes. Subsequently 0.2 ml of contained reaction buffer (10 mM Tris-HCl, 50 mM chloroform was added for each 1 ml of TriPure, and the KCl, 1.5 mM MgCl2, pH 9.0), 0.2 mM of each dNTP, 0.4 tube was vortexed vigorously for 15 sec, incubated at RT mM of each primer and 1.5 U of rTaq DNA polymerase Leiro, J. et al., Leucocyte functions and cytokine responses in turbot scuticociliatosis 59

(Roche). Thermal cycling in an automatic thermal cycler ESP in vitro) than for the TCL cells (prestimulated in vivo (GeneAmp PCR System 2400, Perkin-Elmer, Norwalk, with TCL, presented with TCL in vitro). The specificity USA) was as follows: initial denaturing at 94ºC for 5 of the chemotaxis towards ESP is confirmed by the min; then 35 cycles at 94ºC for 30 sec, 52ºC (TNF-a, marked and significant difference between migration of TGF-b and b-actin) or 45ºC (IL-1b) for 45 sec, and ESP‑prestimulated cells towards ESP and towards L‑15 72ºC for 1 min; and finally a 7-min extension phase at medium alone (Fig. 2). 72ºC. In all experiments we performed controls without RNA or without reverse transcriptase; in no case were amplification products obtained. PCR products (20 ml aliquots) were separated on a 2% agarose gel in TBE buffer stained with 0.5 mg/ml of ethidium bromide, and photographed with a digital camera under a Spectroline 312 variable-intensity UV transilluminator (Spectroline) as previously described (Leiro et al., 2000b). mRNA amounts in each band were quantified using densitometry analysis software (ImageMaster Total Lab, ver. 2.00; AmershamPharmaciaBiotech), and expressed with respect to mRNA amount in the band corresponding to the control gene b-actin, run in the same gel.

2.13. Statistics Data are expressed as means ± SEM. Means were compared (p = 0.05) by one-way ANOVA followed by Tukey-Kramer tests for multiple comparisons.

3. Results 3.1. Leucocyte chemotaxis in response to P. dicentrarchi extracts We first performed in vitro assays to evaluate whether the P. dicentrarchi TCL and ESP preparations are chemoattractants for thioglycollate‑induced turbot peritoneal leucocytes. To this end, groups of 5 turbot were intraperitoneally injected with 1 ml of 3% thioglycollate. On day 3 post‑inoculation, peritoneal leucocytes were obtained, and we then assayed their chemotaxis towards TCL (protein concentration 500 µg/ml), ESP (protein concentration Fig. 1. A: Chemotaxis of peritoneal leucocytes from turbot injected 500 µg/ml), or 10% normal turbot serum (NTS) in L‑15 intraperitoneally with 3% thioglycollate, towards Philasterides medium. The results obtained (Fig. 1A) indicate that both dicentrarchi excretion‑secretion products (ESP), P. dicentrarchi NTS and ESP have significant chemoattractant activity for total lysate (TCL), and normal turbot serum (NTS). Values shown turbot adherent leucocytes. By contrast, migration towards are means ± standard errors (n = 5 fish) of the chemotaxis index (CI, i.e. number of cell migrating towards the candidate attractant, TCL was significantly lower than towards the control (L‑15 divided by number of cells migrating towards medium only in medium alone) (Fig. 1A). About 43% of cells that migrated parallel control assays); asterisks indicate a significant difference towards NTS showed peroxidase activity, versus only about (p < 0.01) with respect to the control. B: Percentage of migratory 30% and 20% of cells that migrated towards ESP and TCL cells (see A) showing peroxidase activity. Values shown are means ± standard errors (n = 5 fish). respectively (Fig. 1B). In a second experiment we inoculated turbot with 3% thioglycollate plus L‑15 medium, ESP (500 µg/ml), or 3.2. Phagocytic activity of inflammatory leucocytes from TCL (500 µg/ml). On day 3 post‑inoculation, peritoneal turbot prestimulated with P. dicentrarchi extracts leucocytes were obtained for assay of chemotaxis towards In this experiment, turbot were inoculated in L‑15 medium, ESP (500 µg/ml), or TCL (500 µg/ml) vivo with 3% thioglycollate containing a) L‑15 medium respectively. The results obtained (Fig.2) indicate that alone, b) L‑15 medium containing ESP (500 µg/ml), or chemotaxis was markedly and significantly higher for the c) L‑15 medium containing TCL (500 µg/ml). On day ESP cells (prestimulated in vivo with ESP, presented with 3 post‑inoculation, peritoneal leucocytes were obtained 60 Leiro, J. et al., Leucocyte functions and cytokine responses in turbot scuticociliatosis and phagocytic activity assayed by incubation with 3.3. ROS production by inflammatory leucocytes in FITC‑labelled K. lactis cells. The results of these assays response to stimulation in vivo and in vitro with P. (Fig. 3) indicate that leucocytes prestimulated with dicentrarchi extracts thioglycollate plus TCL showed significantly greater Groups of 5 turbot were inoculated with 3% phagocytic activity than leucocytes prestimulated with thioglycollate containing L‑15 medium alone, L‑15 thioglycollate plus ESP. medium plus ESP (500 µg/ml), or L‑15 medium plus TCL (500 µg/ml). On day 3 post‑inoculation, peritoneal leucocytes were obtained and stimulated in vitro with PMA (10 µg/ml) or ESP (500 µg/ml), with determination of extracellular ROS production using the fluorogenic R reagent OxyBURST Green H2HFF BSA. In assays in which in vitro stimulation was with PMA, leucocytes prestimulated in vivo with thioglycollate plus TCL showed significantly higher ROS production than leucocytes prestimulated in vivo with thioglycollate plus ESP (Fig. 4, groups B and C). In assays in which in vitro stimulation was with ESP, only leucocytes prestimulated in vivo with thioglycollate plus TCL showed significantly raised ROS production with respect to the control (Fig. 4, group C).

Fig. 2. Chemotaxis of peritoneal leucocytes from turbot injected intraperitoneally with 3% thioglycollate containing L‑15 medium only (group A), P. dicentrarchi excretion‑secretion products (ESP) (groups B and D), or whole P. dicentrarchi extract (TCL) (group C). Leucocytes were obtained 3 days post-inoculation. Candidate chemoattractants were L‑15 medium only (groups A and D), ESP (group B) or TCL (group C). Values shown are means ± standard errors (n = 5 fish) of the chemotaxis index (CI, i.e. number of cell migrating towards the candidate attractant, divided by number of cells migrating towards medium only in parallel control assays); the asterisk indicates a significant difference (p < 0.01) with respect to the control (group A).

Fig. 4. Extracellular reactive oxygen species (ROS) production by turbot adherent peritoneal leucocytes obtained 3 days after intraperitoneal inoculation with 3% thioglycollate plus A) L‑15 medium only, B) P. dicentrarchi excretion‑secretion products (ESP), or C) P. dicentrarchi whole extract (TCL). Leucocytes were stimulated in vitro with ESP or PMA; ROS production was determined as fluorescence emission by

oxidized OxyBURST Green H2HFF BSA (see text); values shown are mean fluorescence increases (arbitrary units) per min, ± standard errors (n = 5 fish); asterisks indicate significant differences (*, p < 0.05; **, p < 0.01) with respect to the control (group A).

3.4. NO production by inflammatory leucocytes in response to stimulation in vivo and in vitro with P. dicentrarchi extracts Fig. 3. Effects of in vivo peritoneal injection of turbot with 3% For assays of NO production, cells were thioglycollate plus P. dicentrarchi excretion‑secretion products (ESP) or total lysate (TCL) on phagocytic activity of adherent prestimulated in vivo by the same procedure as for the peritoneal leucocytes obtained 3 days later. Phagocytic activity assays of ROS production. The cells were then incubated was determined on the basis of ingestion of yeast cells labelled in vitro with LPS or with ESP, with determination of NO with fluorescein isothiocyanate (FITC). Values shown are mean production by the Griess reaction. As shown in Fig. 5, fluorescences (arbitrary units) ± standard errors (n = 5 fish); the asterisk indicates a significant difference (p < 0.01) with respect to NO production was highest by leucocytes prestimulated the control (group A). in vivo with thioglycollate plus ESP, regardless of Leiro, J. et al., Leucocyte functions and cytokine responses in turbot scuticociliatosis 61 in vitro stimulation (Fig. 5, Group B). Leucocytes 3.6. Inflammatory cytokine mRNA levels after in vivo prestimulated in vivo with TCL showed markedly lower stimulation with P. dicentrarchi extracts NO production than leucocytes prestimulated in vivo For this part of the study we determined mRNA with ESP, and indeed than leucocytes prestimulated in levels of the cytokines TNF‑α, IL‑1β and TGF‑β in vivo with thioglycollate only (Fig. 5, Group A versus leucocytes from turbot inoculated with L‑15 medium Groups B and C). only, L‑15 medium containing ESP (500 µg/ml), or L‑15 Independently of the in vivo prestimulation, medium containing TCL (500 µg/ml). Leucocytes were NO production was highest when there was no in vitro obtained 3 days post‑inoculation, and mRNA levels stimulation; in Group B, for example, NO production in estimated by semi‑quantitative RT‑PCR. As shown in Fig. the presence of LPS or ESP was significantly lower than 7, mRNA levels of all three cytokines were significantly in the presence of L‑15 medium only (Fig. 5, Group B, higher in leucocytes from turbot stimulated in vivo with bars 6 and 7 versus bar 5). thioglycollate plus ESP than in leucocytes from the control turbot (stimulated in vivo with thioglycollate only; Fig. 7, Group B). In the case of leucocytes from turbot stimulated in vivo with thioglycollate plus TCL, a) mRNA levels of the anti‑inflammatory cytokine TGF‑β were significantly higher than in both the control turbot and the ESP‑stimulated turbot (Fig. 7, Group C, bar 7); b) mRNA levels of the pro-inflammatory cytokine IL‑1β were significantly lower than in both the control turbot and the ESP‑stimulated turbot (Fig. 7, Group C, bar 8); and c) mRNA levels of the pro-inflammatory cytokine TNF‑α were significantly lower than in the ESP‑stimulated turbot but did not differ significantly from the control turbot (Fig. 7, Group C, bar 9).

Fig. 5. NO• production by turbot adherent peritoneal leucocytes obtained 3 days after intraperitoneal inoculation with 3% thioglycollate plus A) L‑15 medium only, B) P. dicentrarchi excretion‑secretion products (ESP), or C) P. dicentrarchi whole extract (TCL). Leucocytes were stimulated in vitro with ESP or LPS; NO• concentrations (µM) in 48‑hour cultures were determined by the Griess reaction; in some assays N‑monomethyl‑L‑arginine monoacetate (L‑NMMM, 250 µM) was included as a reference inhibitor of NO• production; values shown are mean concentrations (µM) ± standard errors (n = 5 fish); asterisks indicate significant differences (*, p < 0.05; **, p < 0.01) with respect to the groups indicated in brackets; numbers at the base of each bar are group numbers.

3.5. Time‑course of inflammatory cytokine mRNA levels in inflammatory leucocytes To investigate the time‑course of expression of the pro‑inflammatory cytokines TNF‑α and Fig. 6. Time‑courses of mRNA levels of interleukin 1 beta (IL‑1β), IL‑1β, and of the anti‑inflammatory cytokine tumour necrosis factor alpha (TNF‑α) and transforming growth factor TGF‑β, in thioglycollate‑induced leucocytes, we beta (TGF‑β) in peritoneal leucocytes from turbot inoculated 3 days monitored levels of the mRNAs of these cytokines by previously with 3% thioglycollate. Cytokine levels were determined by semiquantitative RT‑PCR. The results shown in Fig. RT‑PCR. A) Agarose gel (2%) analysis of RT‑PCR products using primers for IL‑1β (181 bp), TNF‑α (100 bp), TGF‑β (243 bp) or the housekeeping 6 indicate that mRNA levels of the pro-inflammatory gene β‑actin (330 bp). B) Time‑courses of mRNA levels as determined by cytokines peaked around day 3 post‑inoculation with densitometric analysis [optical density (OD) of cytokine band, divided by thioglycollate, while mRNA levels of TGF‑β peaked OD of band obtained in parallel assay using primer for β‑actin]. Values around day 1. shown are means ± standard errors (n = 5 assays). 62 Leiro, J. et al., Leucocyte functions and cytokine responses in turbot scuticociliatosis

peritoneal leucocytes. Several previous studies have found that parasite extracts induce polarization of fish leucocytes, suggesting the presence of chemoattractants (Taylor & Hoole, 1993). The principal components of parasite excretion‑secretion products typically include proteases, which in P. dicentrarchi show marked cysteine proteinase activity (Paramá et al., 2004). However, the few studies that have been performed to date with proteases secreted by different parasites in human peripheral blood monocytes and neutrophils have indicated that these proteases are involved in inhibition of chemotactic responses and the oxidative burst response (Shepard et al., 1991; Sorensen et al., 1994). The inflamed peritoneal exudates of fish were mainly composed of leucocytes, of which about 32% were neutrophils, about 26% macrophages and about 23% lymphocytes, with only the neutrophils being peroxidase‑positive (Vazzana et al., 2003). Intraperitoneal injection of fish with inflammatory substances augments the proportion of neutrophils (Afonso et al., 1998; Peddie Fig. 7. mRNA levels of interleukin 1 beta (IL‑1β), tumour necrosis et al., 2002). In the present study we observed that factor alpha (TNF‑α) and transforming growth factor beta (TGF‑β) turbot neutrophils showed strong migratory activity in in peritoneal leucocytes from turbot inoculated 3 days previously the absence of serum or other chemoattractants, in line with 3% thioglycollate plus A) L‑15 medium only, B) P. dicentrarchi with Hamdani et al. (1998), while prestimulation in vivo excretion‑secretion products (ESP), or C) P. dicentrarchi whole extract (TCL). Cytokine levels were determined by RT‑PCR. A) Agarose gel with the inflammatory agent thioglycollate increased the (2%) analysis of RT‑PCR products using primers for IL‑1β (181 bp), proportion of neutrophils showing chemotaxis. However, TNF‑α (100 bp), TGF‑β (243 bp) or the housekeeping gene β‑actin the proportion of neutrophils among the migratory cells (330 bp). B) mRNA levels as determined by densitometric analysis declined significantly when parasite extracts (ESP or [optical density (OD) of cytokine band, divided by OD of band obtained in parallel assay using primer for β‑actin]. Values shown are means ± TCL) were offered as candidate chemoattractants. Among standard errors (n = 5 assays); asterisks indicate significant differences the different types of leucocyte present in fish peritoneal (*, p < 0.05; **, p < 0.01) with respect to the groups indicated in exudate (macrophages, neutrophils, eosinophilic granular brackets; numbers at the base of each bar are group numbers.3. cells, lymphocytes, and thrombocytes), only macrophages and neutrophils show significant phagocytic capacity (Do 4. Discussion Vale et al., 2002). Although the viable forms of some Phagocytosis is a complex process with several parasites suppress fish phagocyte function (Leiro et al., steps: chemotaxis, attachment, ingestion, degranulation, 2001; Fast et al., 2002; Gross et al., 2004), extracts of intracellular killing, and intracellular digestion (Bols et these same parasites may stimulate phagocyte function al., 2001). Chemotaxis is a motile behaviour that allows (Scharsack et al., 2003). In the present study, only navigation through different environments, and is widely peritoneal leucocytes from turbot prestimulated in vivo thought to play key roles in parasite virulence: chemotactic with the whole parasite lysate (TCL) showed significantly responses may be involved in tissue identification and increased phagocytic activity, while peritoneal leucocytes penetration, and may facilitate evasion of the host’s from turbot prestimulated with excretion‑secretion immune defences (Lux et al., 2000). In the present study, products (ESP) did not show any increase in phagocytic turbot leucocytes induced in vivo with thioglycollate activity. showed a significant chemotactic response to turbot Depending on the cytokine environment, phagocytic serum (NTS). It is well known that fish serum contains cells (especially macrophages) can differentiate into the anaphylatoxins C3a, C4a and C5a generated during distinct subsets, which can be induced by pro-inflammatory complement activation: these serum components play a microbial molecules (e.g. LPS) or cytokines (e.g. IFN‑γ key role in inflammation (Boshra et al., 2004), and are or TNF‑α) to release inflammatory and/or microbicidal strong chemoattractants and respiratory‑burst‑inducers products, such as ROS (notably the superoxide anion ‑ for fish neutrophils and macrophages/monocytes O2 ), reactive nitrogen species (notably nitric oxide) and (Holland & Lambris, 2004; Rotllant et al., 2004; Boshra pro‑inflammatory cytokines (e.g. TNF,‑ IL 1 and IL‑6). et al., 2004b). In the present study, we found that turbot The persistent presence of these products may result in excretion‑secretion products (ESP), like NTS, acted tissue damage (Noël et al., 2004). Although still rather as powerful chemoattractants for turbot inflammatory poorly understood, the respiratory burst of fish leucocytes Leiro, J. et al., Leucocyte functions and cytokine responses in turbot scuticociliatosis 63

(including macrophages and neutrophils) can be induced study, NO• production was increased in peritoneal by in vitro stimulation with parasite extracts (Taylor & leucocytes prestimulated in vivo with P. dicentrarchi Hoole, 1995; Buchanan & Bresciani, 1999; Muñoz et excretion‑secretion products (ESP), and inhibited in al., 2000). In turbot, inoculation with live P. dicentrarchi leucocytes prestimulated with the whole parasite lysate trophozoites in normal or infected turbot serum (NTS (TCL). This apparent difference in responses to the two or ITS, respectively) leads to significantly increased parasite extracts may be due to the fact that the Griess ‑ ‑ in vitro PMA‑induced extracellular ROS production reaction measures levels of NO2 and NO3 after breakdown by adherent peritoneal leucocytes, as compared with of NO• in aqueous solution (James, 1995); however, if ‑ adherent peritoneal leucocytes from non‑inoculated there is a high concentration of the superoxide anion O2 , ‑ control fish (Leiro et al., 2004). In the present study, in as occurs in cells prestimulated with TCL, the O2 may vivo prestimulation with thioglycollate plus either ESP react with NO• to form peroxynitrite (ONOO), which is or TCL likewise led to significantly increased in vitro not detected in the Griess reaction; in other words, the PMA‑induced extracellular ROS production by adherent apparently low NO• production in TCL‑prestimulated peritoneal leucocytes; however, in vitro ESP‑stimulated cells may be a false negative result. extracellular ROS production was only significantly Previous histological studies have demonstrated increased in leucocytes prestimulated in vivo with that infections of turbot with P. dicentrarchi are followed thioglycollate plus TCL. by marked inflammation of the brain tissues. Inflammation Nitric oxide (NO•) is a signalling molecule with of brain tissues is also common in fish infected with other immunomodulatory capabilities that is generated in ciliates, such as Ichthyophthirius multifiliis (Sigh et al., phagocytic cells from L‑arginine by the Ca2+‑independent 2004). Inflammation is a complex defence mechanism inducible enzyme NO• synthase (iNOS) isoform (Nathan, involved in the destruction of pathogens and other foreign 1992). In mammals, NO• contributes to inflammation bodies: acute inflammation is a generally beneficial (Eisenstein et al., 1994), modulates lymphocyte response, but chronic inflammation may progress to production (Allione et al., 1999) and mediates inflammatory disease (Kaplanski et al. 2003). Cytokines non‑specific anti‑parasite activities (James, 1995). In are a functionally diverse group of cellular messengers fish, the production of NO• by activated macrophages that act through the janus kinase signal transducer and has been demonstrated to occur in response to various activator of transcription (JAK/STAT) and are integral parasite infections (Saeij et al., 2000; Tafalla & Novoa, components of adaptive and innate immune responses 2001; Scharsack et al., 2003). Several aspects of NO• (Alexander & Hilton, 2004). ROS and pro-inflammatory production in fish are controversial: for example, in cytokines act synergically and contribute to the onset and carp the kinetoplastic parasite Trypanoplasma borelli progression of inflammation in various organs (Closa & activates NO• production in vivo, but the related species Folch‑Puy, 2004). In mammals, it has become evident T. carassi does not induce NO• production in vivo, and that interleukin‑1 (IL‑1) and tumour necrosis factor alpha inhibits LPS‑induced NO• production in vitro (Saeij, (TNF‑α), mainly produced by monocytes/macrophages, 2002). Likewise, NO• generation after stimulation with are the principal mediators of tissue destruction in many LPS appears to vary among fish species: LPS induces immune‑inflammatory diseases (Dayer & Burger 1994). In NO• production by goldfish and carp leucocytes (Stafford parasite infections by monogeneans it has been proposed et al., 2002; Saeij et al., 2003; Stafford et al., 2004), but that the release of cytokines (e.g. IL‑1, TNF and IFN) not by sea bass head‑kidney macrophages (Sarento et al., initiates a series of events leading to a decrease in the 2004). In turbot, two different macrophage populations ectoparasite population (Buchmann, 1999), and the pro- have been identified, one producing NO in response to inflammatory cytokine IL‑1β has been postulated as an LPS and the other non‑responsive to LPS alone, though immune adjuvant (Ying & Kwang, 2000). In rainbow trout, responding to LPS plus turbot macrophage activating Oncorhynchus mykiss, the pro-inflammatory cytokine actor (MAF) or LPS plus normal turbot serum (Tafalla IL‑1β is specifically expressed during infection with the & Novoa, 2000; Leiro et al., 2004). In rainbow trout it monogenean Gyrodactylus derjavini (Lindenstrom et has also been shown that LPS does not induce expression al., 2003). In this fish species, release of TNF has also of the interferon regulatory factor 1 (IRF‑1) gene, which been seen during primary infections with G. derjavini stimulates the promoters of various IFN‑induced genes (Lindenstrom et al., 2004), and during a natural outbreak (Collet & Secombes, 2002) and which, together with TNF, of proliferative kidney disease (PD) due to a myxozoan stimulates NO• production (Noël et al., 2004). Infection parasite (Holland et al., 2003). Carp intraperitoneally of turbot with viable trophozoites of P. dicentrarchi injected with the flagellate parasite Trypanoplasma leads to increased NO• production by adherent peritoneal borelli up‑regulate expression of the TNF‑α and IL‑1β leucocytes stimulated with LPS and NTS or infected genes (Saeij et al., 2003a); however, during infection with turbot serum (ITS) (Leiro et al., 2004). In the present the bacteria Renibacterium salmonarum, macrophages 64 Leiro, J. et al., Leucocyte functions and cytokine responses in turbot scuticociliatosis showed a rapid inflammatory response in which the (Saeij et al., 2003a), while rainbow trout infected with the expression of IL‑1β was enhanced but TNF‑α expression ciliate Ichthyophthirius multifiliis show increased IL‑1β was greatly reduced (Grayson et al., 2002), indicating a and TNF‑α expression by day 4 after infection (Sigh et possible mechanism for modulation of the host immune al, 2004). response by the parasite. Transforming growth factor beta In conclusion, the results of the present study (TGF‑β) is an immunoregulatory cytokine with primarily suggest that the immunopathogenic effects of Philasterides suppressive properties, including down‑regulation of dicentrarchi infection are attributable to the parasite’s MHC expression, production of some cytokines, T and B excretion‑secretion products. These effects may reflect cell proliferation, IgG and IgM production, IL‑2 receptor over‑expression of proinflammatory cytokines and expression, cytotoxic T‑cell generation and function, • ‑ over‑production of NO and O2­ by phagocytic cells. Under LK and NK cell activation and function, macrophage this hypothesis, as parasites are destroyed by the immune activation, and macrophage respiratory burst activity system, there will be an increase in the endocytic activity (Ruscetti & Palladino, 1991). However, TGF‑β has of phagocytic cells, and up-regulation of the expression some pro‑inflammatory effects, including promotion of of TGF‑β, which has been shown to play a role in ablating macrophage and neutrophil chemotaxis, IgA production, of iNOS and thus inhibition of NO production (Leiro et and production of some cytokines (Harms et al., 2003). al., 2003); these processes would thus tend to block the Whether TGF‑β has pro‑ or anti‑inflammatory effects acute inflammatory response to the infection. depends on its concentration, the differentiation status of the target cells, and the concentration of other 5. Acknowledgements pro‑inflammatory compounds; In any case, it plays a This work was supported by grants critical immunoregulatory role by limiting damage to PGIDIT02RMA23701PR from the Xunta de Galicia (Spain) tissues during resolution of the normal immune response and AGL2003-04644 from the Comisión Interministerial de (McCartney‑Francis & Wahl, 1994). TGF‑β isoforms Ciencia y Tecnología (CICYT), Spain. (TGF‑β1, β2 and β3) have been described in many fish species (Tafalla et al., 2003). The present study clearly 6. References shows that transcription of the IL‑1β, TNF‑α and TGF‑β Afonso, A.; Lousada, S.; Silva, J.; Ellis, A.E.and Silva, M.T. genes occurs in thioglycollate‑induced turbot peritoneal 1998. Neutrophil and macrophage responses to leucocytes, peak levels of IL‑1β mRNA and TNF‑α inflammation in the peritoneal cavity of rainbow trout mRNA appearing around 3 days after inoculation with Oncorhynchus mykiss. A light and electron microscopic thioglycollate, or around 1 day post‑inoculation in the cytochemical study. Dis Aquat Organ, 34, 27-37. case of TGF‑β. Transcription of the IL‑1β gene began Alexander, W.S. and Hilton, D.J. 2004. The role of suppressors within 24 hours; however, transcription of the TNF‑α of cytokine signaling (SOCS) proteins in regulation of gene was not detected during this period. In the sea bass the immune response. Ann Rev Immunol, 22, 503-529. Dicentrarchus labrax, transcription of the IL‑1β gene was Allione, A.; Bernabei, P.; Bosticardo, M.; Ariotti, S.; Forni, G. likewise detected within 24 hours of in vivo stimulation and Novelli, F. 1999. Nitric oxide suppresses human T lymphocyte proliferation through IFN-γ-dependent and with LPS (Scapigliati et al., 2001). In the present study, IFN-γ-independent induction of apoptosis. J Immunol, mRNA levels of all three cytokines declined over time, 163, 4182-4191. in each case dropping significantly below the peak Baggiolini, M. and Wymann, M.P. 1990. Turning on the level by day 7 post‑inoculation. When turbot were respiratory burst. Trends Biochem Sci, 15, 69-72 inoculated in vivo with thioglycollate plus P. dicentrarchi Bols, N.C.; Brubacher, J.L.; Ganassin, R.C. and Lee, L.E. 2001. excretion‑secretion products (ESP), mRNA levels of all Ecotoxicology and innate immunity in fish. Dev Comp three cytokines reached significantly higher levels than Immunol, 25, 853-873. in control turbot inoculated with thioglycollate only. Boshra, H.; Li, J.; Peters, R.; Hansen, J.; Matlapudi, A. and When turbot were inoculated in vivo with thioglycollate Sunyer, J.O. 2004a. Cloning, expression, cellular plus the P. dicentrarchi whole extract (TCL), mRNA distribution, and role in chemotaxis of a c5a receptor in levels of IL‑1β and TNF‑α were close to the levels seen rainbow trout: the first identification of a c5a receptor in a nonmammalian species. J Immunol, 172, 4381-4390. in control turbot, while mRNA levels of TGF‑β were Boshra, H.; Peters, R.; Li, J. and Sunyer, J.O. 2004b. Production significantly higher than in the ESP‑stimulated turbot. of recombinant C5a from rainbow trout (Oncorhynchus Increased expression of the TNF‑α gene has similarly mykiss): role in leucocyte chemotaxis and respiratory been observed in a previous study of rainbow trout burst. 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