[show abstract][hide abstract] ABSTRACT: Glucocorticoid actions on the immune system are diverse and cell type dependent, and little is known about cell type-specific interactions and cross-talk between hormones and cytokines. In this study we have analyzed the gene expression patterns of the rainbow trout macrophage cell line RTS-11 by quantitative PCR, after exposure to combinations of cortisol plus a pro-inflammatory cytokine (e.g. recombinant trout IL-1β, IFN-γ), type I IFN or a PAMP (LPS or poly I:C). Several key genes of the inflammatory process were targetted to assess whether any modulation of their expression occurred due to the addition of cortisol to this cell line. Incubation of macrophages for 3 or 6 h with a physiological concentration of cortisol caused a decrease in expression of IL-6 and IL-8, but no significant changes were observed for the other genes examined. Co-stimulation of cortisol with the inflammatory agents resulted in a general suppression of genes related to the inflammatory response. Cortisol inhibited the up-regulation of IL-8 by all the stimulants after 3 h of co-incubation. Suppression of the up-regulation of IL-6 by rIL-1β, rIFN-γ and poly I:C, of γIP by rIFN-γ or poly I:C, and of Cox-2 by rIL-1β was seen after 6 h. In contrast, cortisol in combination with the pro-inflammatory agents has a synergistic effect on IL-10 expression, an anti-inflammatory molecule, suggesting that the activation of certain macrophage functions that lead to the resolution of inflammation occurs in fish macrophages in response to cortisol treatment.
Fish & Shellfish Immunology 10/2010; 30(1):215-23. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: An interferon (IFN)-gamma responsive stable cell line RTG-3F7 has been developed for rainbow trout by modifying the RTG-2 cell line through transfection with a plasmid construct (pGL4.14[luc2/hygro]-PrTAP2) containing a promoter element from the IFN-gamma responsive gene TAP2 linked to a luciferase reporter gene and a hygromycin resistance gene. Following transfection single clones were selected in 96 well plates using hygromycin B, and those showing specific activation after rIFN-gamma stimulation were maintained. Five clones that showed the highest reporter activity to rIFN-gamma were incubated with different stimuli to examine specificity. No significant induction of luciferase was observed following exposure to recombinant type I IFN, LPS, PHA or poly I:C. The cell line was responsive to rIFN-gamma at concentrations between 150 pg and 20 ng ml(-1). Supernatants of primary cultures of head kidney leucocytes stimulated with PHA, known to induce IFN-gamma gene expression, were also used to assess the reporter activity of the stable cell line. A dose-dependent induction of the promoter activity was observed with these supernatants indicating the presence of IFN-gamma. These results indicate that the stable cell line RTG-3F7 is an excellent tool for monitoring the presence of trout IFN-gamma in biological samples, and in addition, enables the study of intracellular signalling pathways of IFNs, their receptor interactions, and other closely related signalling networks.
Fish & Shellfish Immunology 11/2009; 28(2):312-9. · 2.96 Impact Factor
[show abstract][hide abstract] 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 RESV on 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 E(2) (PGE(2)) 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.
Veterinary Immunology and Immunopathology 11/2008; 126(1-2):9-19. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: Reporter constructs of three interferon (IFN)-gamma-induced rainbow trout genes were generated to examine specificity to type I or type II IFN. Constructs included gammaIP-10, LMP2 and TAP2 and were used to transfect trout fibroblast cells (RTG-2) which were then exposed to rainbow trout rIFNs. The gammaIP-10 construct showed high reporter activity even in the absence of rIFNs. The LMP2 promoter contained one GAS element and two double ISRE elements, of four constructs made, only those with ISRE elements showed significant reporter activity following rIFN-gamma stimulation. The TAP2 regulatory region contained two GAS, two ISRE and one C/EBP element from which four constructs were made. Reporter expression for the construct containing all five elements showed an 11- and 2-fold increase in response to rIFN-gamma and type I rIFN, respectively. Constructs containing only the GAS elements did not respond to rIFNs. The TAP2 construct with two ISRE and the C/EBP gave the greatest dose-dependent reporter response to rIFN-gamma, with no significant response to type I rIFN. These data suggest that the ISRE elements, or elements nearby, are essential for the induction of type II IFN responsive genes in trout. The TAP2 construct is a candidate to develop a IFN-gamma reporter stable cell line.
[show abstract][hide abstract] ABSTRACT: The protection induced in turbot by inactivated vaccines containing either of two isolates (I(1) and C(1)) of the scuticociliate parasite Philasterides dicentrarchi, which causes important mortalities in turbot cultures, was evaluated in the present study. The results obtained after challenging the fish with the two isolates show that vaccination protected fish only against the homologous isolate, but did not confer cross-protection. The two isolates constitute two serotypes, as shown in the immobilization tests with mouse and turbot anti-I(1) and anti-C(1) antisera, in which only the homologous antisera immobilized the ciliates. ELISA assays, using total antigen free of proteases (TAWP), cytosolic antigens (CYA), ciliar antigens (CA) or membrane protein fraction (MPF), were also carried out. Differences in the levels of antibodies produced in mouse against the homologous and heterologous antigens were observed; these differences were significantly different when the antigen preparations used in the ELISA were TAWP, CYA or CA. Nevertheless, ELISA assays using turbot sera against TAWP did not show significant differences in the levels of antibodies against the homologous and heterologous antigens. Antigenic cross-reactivity was also detected in the Western blot assays, as well as significant differences in the patterns of antigenic recognition in the two isolates - in both reduced and non-reduced TAWP antigens, but which was noteworthy when mouse antisera were used. The results obtained in the present study demonstrate for the first time the existence of serotypes of the ciliate parasite of turbot Philasterides dicentrarchi that display clear antigenic differences, which must be taken into consideration in the future development of a vaccine against scuticociliatosis.
Fish & Shellfish Immunology 07/2008; 25(4):417-24. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: The efficacy of a vaccine against the fish pathogen Philasterides dicentrarchi was evaluated in turbot by measuring the production of specific antibodies and duration of protection. Four groups of turbot were vaccinated twice, on days 0 and 30, with phosphate-buffered saline, mineral oil adjuvant, antigen or antigen plus adjuvant. Specific serum antibodies were determined on day 0 and 1 month after the first and the second vaccinations. Protection was evaluated 1 month after the first vaccination and 1 and 5 months after the second vaccination. Serum antibody titres, measured by enzyme-linked immunosorbent assay, and protection, assessed by challenges, increased significantly 1 month after the second vaccination in the group injected with antigen plus adjuvant and the protection lasted for at least a further 5 months in this group. The relative protection was 77% and 66% 1 and 5 months after the second vaccination, respectively. Administration of antigen or adjuvant separately had no effect on antibody response or protection. The results indicate that emulsion containing antigen plus adjuvant induced durable protection against P. dicentrarchi after the administration of the two vaccinations, and that this preparation can be used as a vaccine against the pathogen.
Journal of Fish Diseases 03/2008; 31(2):135-40. · 1.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Myeloperoxidase (MPO) is a conspicuous enzyme in neutrophils of many fish species. Although the MPO gene has been identified in some fish species, the structure and functions of the protein remain to be determined in these vertebrates. In the present study, we isolated turbot neutrophil MPO from kidney cells by affinity chromatography, with Ulva rigida acidic sulphated polysaccharides (ASP), some of which resemble glycosaminoglycans, and Sepharose. The product obtained, of approximately 150kDa molecular weight and with peroxidase activity, was examined by SDS-page electrophoresis under reduced conditions and immunoblotting, and a single band of about 75kDa was observed. The results obtained suggest that turbot MPO is a dimer and that the band of 75kDa probably corresponds to a monomer generated by treatment of the samples with the reducing agent. The band was analysed by electromatrix-assisted laser desorption ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization-ion trap mass spectrometry, dynamic exclusion mode (LC-ESI-IT DE), to determine the amino acid composition of some peptides. The peptides obtained were very similar to myeloperoxidases of other organisms, including other fish and mammals, and were used to design the primers for cDNA amplification. A 567bp product was amplified and the deduced amino acid sequence, which contains several putative N-glycosylation and O-glycosylation sites, was compared with other myeloperoxidases. As expected, turbot MPO was more similar to MPO from other fish species (67-86% identity), where the phylogenetic tree obtained agrees with the taxonomic hierarchy, than to MPO from mammals (55-57% identity) and other groups. The results obtained in the present study will also allow functional studies to be carried out with turbot neutrophil MPO enzyme, as well as analysis of MPO gene expression under different stimuli.
[show abstract][hide abstract] ABSTRACT: Philasterides dicentrarchi is a scuticociliate parasite that causes important mortalities in cultured turbot and therefore considerable economic losses. In the present study, we applied for the first time a factorial experimental design to optimize the formulation of an inactivated anti-P. dicentrarchi vaccine on the basis of three of its main components: antigenic dose, concentration of inactivating agent and proportion of adjuvant in the emulsion, and determined the effect of each factor on protection against the parasite and on antibody production. We found that concentration of antigen was the only one of the three factors considered that had a significant effect on the level of specific serum antibodies. In addition, we showed a statistically significant relationship between the level of antibodies and the mean time of survival of the vaccinated fish. The vaccine formulation containing 106 trophozoites/ml, 0.2% formalin and 50% adjuvant generated the longest mean time of survival, 100% protection and the highest levels of antibodies in the vaccinated fish.
[show abstract][hide abstract] ABSTRACT: The effects exerted by cysteine proteinases isolated from the histiophagous ciliate Philasterides dicentrarchi on the phagocytic functions of turbot pronephric leucocytes (PL) were investigated. The enzymes were tested at concentrations of 125, 250 and 500 microg ml(-1), and it was found that the viability of the leucocytes was not affected after treatment for 24h. Leucocyte migration was inhibited by the cysteine proteinases in a dose-dependent manner, whereas the ascitic fluid obtained from turbot experimentally infected with P. dicentrarchi induced high chemotactic activity in the turbot PL. The proteinases did not affect yeast cell phagocytosis but increased intracellular production of the superoxide anion (O2(-)). Stimulation with the proteinases did not alter the PGE2 levels in supernatants from 24-h cultures of PL, however, beta-glucans (100 microg ml(-1)) provoked a large increase in PGE2 levels, which were inhibited after addition of 10 microg ml(-1) of indomethacin, a non-selective inhibitor of COX2 enzymatic activity. The mean PGE2 level in ascitic fluid from turbot, experimentally infected with P. dicentrarchi, was 500 pg ml(-1), and the addition of low levels of PGE2 (62.5 pg ml(-1)) to PL cultures stimulated O2(-) production, although addition of PGE2 at concentrations higher than 250 pg ml(-1) blocked the increase in stimulation. Addition of cysteine proteinases to 24-h cultures of PL also increased mRNA levels in the pro-inflammatory cytokine interleukin-1beta. The results revealed the capacity of cysteine proteinases isolated from P. dicentrarchi to modulate the innate immune response of turbot, which together with the inflammation mediators produced during infection, may play an important role in pathogenesis of the disease and in the survival of the parasite.
Fish & Shellfish Immunology 12/2007; 23(5):945-56. · 2.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Water-soluble acidic polysaccharides from the cell walls of Ulva rigida are mainly composed of disaccharides that contain glucuronic acid and sulphated rhamnose. The structure of disaccharides resembles that of glycosaminoglycans (GAGs) as they both contain glucuronic acid and sulphated sugars. Glycosaminoglycans occur in the extracellular matrix of animal connective tissues but can also be produced by leucocytes at inflammatory sites. Certain types of GAGs can even activate macrophages and therefore the acidic polysaccharides from U. rigida probably modulate macrophage activity. In the present study, we evaluated the effects of U. rigida polysaccharides on several RAW264.7 murine macrophage activities, including expression of inflammatory cytokines and receptors, nitric oxide and prostaglandin E2 (PGE(2)) 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 several chemokines (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 of macrophages with U. rigida polysaccharides also induced an increase in nitrite production, although this effect decreased considerably after desulphation of polysaccharides, suggesting that the sulphate group is important for the stimulatory capacity of these molecules. U. rigida polysaccharides also stimulated macrophage secretion of PGE(2) and induced an increase in COX-2 and NOS-2 expression. The results indicate that U. rigida acid polysaccharide can be used as an experimental immunostimulant for analysing inflammatory responses related to macrophage functions. In addition, these polysaccharides may also be of clinical interest for modifying certain macrophage activities in diseases where macrophage function is impaired or needs to be boosted.
International Immunopharmacology 08/2007; 7(7):879-88. · 2.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: The role of proteinases of the histiophagous ciliate Philasterides dicentrarchi, purified by affinity chromatography in bacitracin-Sepharose, on apoptosis (programmed cell death) of turbot pronephric leucocytes (PL) was investigated. The results showed that more than 90% of proteinases purified by bacitracin-Sepharose were cysteine proteinases, which lacked significant caspase-3-like activity and generated three main gelatinolytic bands of molecular weights 36, 45 and 77 kDa as determined by gelatine-SDS-PAGE and immunoblot. Viability of PL cells after 24 h stimulation with P. dicentrarchi cysteine proteinases did not differ from that of non-stimulated cells. Apoptosis was confirmed by: (i) caspase activity, (ii) DNA fragmentation, and (iii) nucleus fragmentation. The caspase-3-like activity in PL incubated for 4h in the presence of 125, 250 and 500 microg/ml of proteinases increased in a dose-dependent fashion. The PL DNA was fragmented following 24-h exposure to P. dicentrarchi cysteine proteinases and characteristic DNA ladders consisting of multimers of approximately 180-200 pb were produced. Morphological changes, such as chromatin condensation and nucleus fragmentation, were observed under fluorescence microscopy after DAPI staining of the PL cells incubated with cysteine proteinase-incubated for 24 h. The results suggest that the pathogenic scuticociliate P. dicentrarchi may induce host leucocyte programmed cell death via the production of cysteine proteinases, as a mechanism of pathogenesis and evasion of the turbot innate immune response.
International Journal for Parasitology 02/2007; 37(1):87-95. · 3.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: We previously reported that Ulva rigida polysaccharides enhance the respiratory burst activity of turbot kidney leucocytes. In the present study, water-soluble polysaccharides were separated into several components to determine the relationship between their structure and their effects on cell activity. U. rigida polysaccharides were found to be composed mainly of uronic acids, rhamnose and xylose with smaller amounts of other sugars. Anion exchange chromatography was used to separate the polysaccharides into acid (94%) and neutral sugars (6%). U. rigida polysaccharides induced respiratory burst activity of turbot kidney leucocytes and the effect was found to be related to the acid fraction. The acid fraction was separated into four fractions, according to the molecular size, and all had stimulatory capacity. Acid sugars contained mainly large homogeneous sulphated polysaccharides. The capacity to induce the respiratory burst was lost after desulphation of the polysaccharides, but was recovered again after non-specific resulphation. The acid polysaccharides induced also IL-1β expression in turbot peritoneal leucocytes. As above, this effect was lost after desulphation of the polysaccharides. The present results indicate that the sulphate groups are required for induction of turbot innate immune responses by U. rigida polysaccharides.
[show abstract][hide abstract] ABSTRACT: In this study, we investigated the effects of water-soluble extracts (WSE) from seaweed species Ulva rigida C. Agardh, Enteromorpha sp., Codium tomentosum (Huds) Stackh., Fucus vesiculosus L., Pelvetia canaliculata (L.) Decne et Thur, Dictyota dichotoma (Huds) Lamour, Chondrus crispus Stackh and Porphyra umbilicalis (L.) J. Agardh, on the respiratory burst of turbot phagocytes, in search of biologically active substances with immunostimulating capacities. The stimulatory capacities of the extracts varied greatly, depending on their origin, the concentrations used and the time of incubation. The best responses were induced by the extracts obtained from U. rigida, Enteromorpha sp. and C. crispus. Pre-incubation of the phagocyte cells with U. rigida and C. crispus extracts and subsequent incubation with phorbol myristate acetate (PMA) showed that these cells responded to further stimulation by this substance and, at some concentrations, they showed higher respiratory burst activity than control cells stimulated by PMA alone, suggesting that the extracts had a priming effect. We also tested the effects of protein-free water-soluble extracts (PF-WSE) and of the polysaccharide fractions (PSF) obtained from the PF-WSE of U. rigida and C. crispus. These extracts induced an increase in the respiratory burst activity of turbot phagocytes, suggesting that most of the stimulatory capacity of the water-soluble extracts was associated with polysaccharides.
[show abstract][hide abstract] ABSTRACT: The present study evaluated the effect of oral administration of three types of glucan on the resistance of gilthead seabream to Photobacterium damselae subsp. piscicida and on the activity of its phagocytes. Groups of fish were fed with a basal diet (controls) or with the basal diet supplemented with glucan for two different periods of time before being bath challenged with the bacterium. Groups of fish fed with 1 and 10 g kg−1 of glucan for a short period (2 weeks with glucan and 1 week with the basal diet) showed a higher degree of protection against pasteurellosis than the control group. This was especially pronounced in groups of fish fed with the higher concentrations of glucan. The respiratory burst and phagocytic activity of spleen phagocytes varied with time but no significant differences were found between groups in the third week when the challenge was carried out. The group of fish fed with 1 g kg−1 of glucan for longer periods of time (2 weeks with glucan, 1 week with the basal diet, 2 weeks with glucan, and 1 week with the basal diet) also showed enhanced protection against P. damselae. However, the group fed with 5 g kg−1 had mortality rates comparable to the control group, and protection in the group fed with 10 g kg−1 of glucan was significantly lower than in the control group. These results suggest that glucans can be used in the diet to prevent or reduce mortalities in gilthead seabream due to pasteurellosis, and they show the importance of the concentration and the period of administration of glucan to obtain optimal protection against this disease.
[show abstract][hide abstract] ABSTRACT: Monocytes/macrophages obtained from the head kidney and peritoneal cavity of gilthead seabream (Sparus aurata) were cultured using plates from three different manufacturers, and were maintained under different conditions. The effects on the morphology and fusion of monocytes/macrophages of initial cell loading, removal of non-adherent cells at different times after plating, and addition of serum and antibiotics were evaluated by light microscopy, and transmission (TEM) and scanning (SEM) electron microscopy. Despite variations in adherence, the behaviour and the morphological changes in kidney monocytes/macrophages were similar in all three types of plates. When foetal calf serum (FCS) was added to the incubation medium, most of the cells resembling monocytes/macrophages were connected by cytoplasmic extensions that formed bridges after 24 hr in culture. After 30 hr, the monocytes/macrophages started to fuse, forming multinucleated giant cells (MGCs) which gradually increased in size until the culture was 4-5 days old. After 5 days the MGCs started to die, and after a week most had disappeared from the cultures. Cells incubated with medium without serum showed changes similar to those fed with FCS, but some cells survived for 3 weeks. The addition of fish serum to the medium appeared to accelerate all processes: the monocytes/macrophages and MGCs died after 3 days in culture. Antibiotics had no apparent effect on the cultures. Removal of non-adherent cells at different times after plating did not appear to affect cell fusion. Coating the wells with extracellular matrix proteins reduced adherence but did not inhibit cell fusion. Curiously, not all macrophages fused with MGCs, and, unlike MGCs, these macrophages phagocytosed sheep red blood cells (SRBCs). Peritoneal macrophages also fused and formed MGCs in culture, similarly to kidney cells.
[show abstract][hide abstract] ABSTRACT: Here we show, by spectrophotometry and enzyme cytochemical methods, that turbot neutrophils and gilthead seabream acidophils responded in a similar way when incubated with PMA or with particulate glucans. Cells stimulated with PMA released high amounts of superoxide both intra- and extracellularly. However, O2- was mainly released intracellularly when cells were incubated with particulate glucans. Small glucan particles were quickly phagocytosed and O2- was initially produced in intracellular vesicles and tubular structures that later fused with the phagosome or with the cell membrane. Large glucan filaments that were not phagocytosed also induced cell stimulation and O2- was also produced in intracellular vesicles which then appeared to fuse with the cell membrane. We conclude that, in stimulated turbot neutrophils and gilthead seabream acidophils, superoxide production is carried out initially in intracellular compartments that are very similar to those described in mammalian neutrophils.
[show abstract][hide abstract] ABSTRACT: The in vitro effect of several β-glucans on the respiratory burst of turbot and gilthead seabream phagocytes was examined. Three particulated β-glucans from yeast, Saccharomyces cerevisiae and a particulate glucan from the fungus Schizophyllum commune were used. In some experiments, cells were incubated for 1 or 2 h with a mixture of glucan (0–500 μg ml−1) and nitroblue tetrazolium (NBT). In others, cells were preincubated with glucans for 1, 3 and 6 h and then incubated for 1 h with NBT with or without PMA. Cells from gilthead seabream and turbot responded similarly to glucans, and differences in activity depended mainly on the concentration of glucans, the length of incubation period of cells and glucan, and on the glucan used. Incubation of cells with glucans for 1 h directly induced a respiratory burst which increased with the concentration of glucan. However, after 2 h incubation a decrease in NBT reduction occurred at the highest glucan concentrations. An enhancement of the respiratory burst, which increased with the concentration of glucan, was also seen when cells were preincubated with glucans and then incubated with NBT without PMA. However, when PMA was added to the NBT solution, the highest NBT reduction was found at low glucan concentrations whereas with higher concentrations of glucan the NBT reduction decreased significantly. Thus high concentrations of glucan directly induced respiratory burst and led to exhaustion. Low concentrations of glucan primed the phagocytes to be capable of enhanced production of reactive oxygen species on subsequent activation of the respiratory burst. The former may increase disease susceptibility, the latter increase resistance.