Fish & Shellfish Immunology (2002) 13, 279–291 doi:10.1006/fsim.2001.0403 Available online at http://www.idealibrary.com on
Changes in some innate defence parameters of seabream (Sparus aurata L.) induced by retinol acetate A. CUESTA, J. ORTUNx O, A. RODRIGUEZ, M. A. ESTEBAN AND J. MESEGUER* Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain (Received 9 October 2001, accepted 10 December 2001, published electronically) The e#ects of high doses of dietary or intraperitoneally (i.p.) injected retinol acetate on the gilthead seabream (Sparus aurata L.) innate immune system were studied. Gilthead seabream specimens were fed a commercial nonsupplemented diet containing 1·75 mg of vitamin A kg 1 (as control) or the same diet supplemented with 50, 150 or 300 mg of retinol acetate kg 1 (as vitamin A source). After 1, 2, 4 or 6 weeks, serum samples and head-kidney leucocytes were obtained from each fish. Serum lysozyme activity and myeloperoxidase (MPO) content were una#ected by the vitamin A diet content. The phagocytic and respiratory burst activities of head-kidney leucocytes were established, as well as their myeloperoxidase content. While phagocytosis was not enhanced by dietary vitamin A intake and was even slightly decreased after 2 weeks, respiratory burst activity was enhanced in specimens fed supplements of 150 and 300 mg retinol acetate kg 1 diet for 1 or 2 weeks. Leucocyte MPO content was also enhanced when seabream were fed the highest vitamin A dose for 2 or 4 weeks and after being fed the 150 or 50 mg supplemented diets for 4 or 6 weeks, respectively. Three di#erent groups of seabream were i.p. injected with 1 ml of phosphate bu#er containing an amount of retinol acetate equivalent to the daily dietary supplements from the first experiment (0-control-, 0·05 or 0·30 mg 100 g 1 biomass). Both injection doses of retinol acetate were toxic for the gilthead seabream which showed hypervitaminic e#ects. These data show that retinol acetate plays an important role in the gilthead seabream nonspecific cellular immune system due to its antioxidant properties. They also point to the importance of the way in which it is administered, by dietary uptake or intraperitoneal injection. 2002 Elsevier Science Ltd. All rights reserved.
Key words:
retinol acetate, vitamin A, lysozyme activity, phagocytosis, respiratory burst, myeloperoxidase, gilthead seabream (Sparus aurata L.).
I. Introduction The naturally occurring compounds provitamin A (carotenoids) and vitamin A (retinol and its derivatives) have been related to growth, cellular di#erentiation and cell–cell or cell–substrate interactions [1]. In mammals, *Corresponding author. E-mail:
[email protected] 1050–4648/02/$-see front matter
279 2002 Elsevier Science Ltd. All rights reserved.
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hypovitaminosis A is associated with the impairment of linear growth, cartilage and bone development, changes in epithelial cell di#erentiation and function, xerophthalmia and blindness [2]. Moreover, vitamin A has been widely related to the immune system of this vertebrate group (reviewed by Ross [1] and Bendich & Olsen [3]). In mammals, vitamin A displays potent anti-tumour activity and slightly enhances phagocytic functions [4–10]. It also enhances antibody responses [11] and lysozyme activity [12]. The possible mechanisms involved in the mode of action of vitamin A remain unresolved although anti-oxidant and adjuvant properties may be related. In teleosts, as in other animals, vitamin A is incorporated directly from the diet or metabolised from carotenoids [13]. This makes it an essential micronutrient for fish. Increased disease resistance of salmonids fed vitamin A has been reported [14]. However, despite these preliminary results and the great interest that vitamin A (and other vitamins) might have on the fish immune system, there are very few studies of the immunomodulatory e#ect of this vitamin in fish [15–17]. Furthermore, the available data are controversial and, to our knowledge, only one paper describes a clearly positive e#ect of retinol acetate on the fish immune system or, more specifically, on the natural cytotoxic activity of channel catfish [15]. The mortality rate of Atlantic salmon specimens injected with Aeromonas salmonicida was lower in fish which had been treated for 4 months with retinol acetate than in fish fed a reduced vitamin A diet, although no positive e#ect upon their humoral immune response or upon leucocyte phagocytosis was observed [16]. Since vitamin A may act as a possible immunostimulant and be of potential use in fish-farming, the aim of the present work was to study the e#ects of high retinol acetate dosages administered in the diet or by intraperitoneal injection on certain innate immune parameters of gilthead seabream (Sparus aurata L.). II. Materials and Methods ANIMALS
One hundred and seventy specimens (150 g mean weight) of the hermaphroditic protandrous seawater teleost gilthead seabream (Sparus aurata L.) obtained from Culmarex S.A. (Murcia, Spain) were kept in 450 l glass fibre, running seawater aquaria, 28‰ salinity, at 20 C and with a 12 h light:12 h dark photoperiod.
RETINOL ACETATE SUPPLEMENTED DIETS
Four experimental diets were prepared in the laboratory from a commercial pellet diet (Trouw Espan˜a, Burgos, Spain) (vitamin A content 1·75 mg kg 1 diet). For this, three solutions of 2, 6 and 12 mg all-trans-retinol acetate (500 000 USP units g 1, Sigma) ml 1 fish oil were made. The supplemented diets were prepared daily by spraying the vitamin solutions uniformly on the feed at a ratio of 25 ml kg 1 dry weight to obtain supplementations of 50, 150 and 300 mg retinol acetate kg 1. The non-supplemented diet (control) was
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sprayed with fish oil only. As determined by a high-pressure-liquidchromatography (HPLC) method [16] the all-trans-retinol concentration in the fish oil was 65 g ml 1. One hundred specimens were divided randomly into four groups, which were distributed into four aquaria, and each group was fed one of the four di#erent diets. Fish were fed at a rate of 10 g dry diet kg 1 biomass (1%) per day. The biomass in each aquarium was measured before the experiment and the daily ration was adjusted accordingly after each sampling. SAMPLE COLLECTION
Six fish of each aquarium were randomly sampled after 1, 2, 4 and 6 weeks of treatment. The specimens were anaesthetised with benzocaine (4% in acetone) (Sigma), weighed and measured. Blood samples were collected from the caudal vein and allowed to clot at room temperature for 4 h. After centrifugation, serum was removed and frozen at 80 C until the determination of lysozyme activity and myeloperoxidase content. Head-kidney leucocytes were isolated from each specimen under sterile conditions. Briefly, head-kidney was excised, cut into small fragments and transferred to 8 ml of supplemented sRPMI-1640 [RPMI-1640 culture medium (Gibco) with 0·35% sodium chloride (to adjust the medium’s osmolarity to gilthead seabream plasma osmolarity, 353·33 mOs), 100 iu ml 1 penicillin (Flow), 100 g ml 1 streptomycin (Flow) and 10 iu ml 1 heparin (Sigma)]. Cell suspensions were obtained by forcing fragments of the organ through a 102 m nylon mesh. Head-kidney cell suspensions were layered over a 48% Percoll density gradient (Pharmacia) and centrifuged at 400g for 30 min at 4 C [18]. After centrifugation, the band of leucocytes above the 48% interface was collected with a Pasteur pipette and washed twice. Cell viability was higher than 98%, as determined by the trypan blue exclusion test. GROWTH
After each sampling, specific growth rate (SGR, % body weight/day) for each group was determined using the equation SGR=100 (logn Wf logn Wo)/t, where Wo and Wf were the initial and final weights of each experimental group, respectively, after t days [19]. LYSOZYME ACTIVITY
Lysozyme activity was measured according to the turbidimetric method described by Parry et al. [20]. The lysozyme substrate was a 0·75 mg ml 1 lyophilised Micrococcus lysodeikticus (Sigma) suspension in 0·1 M sodium phosphate/citric acid bu#er, pH 5·8. Serum (25 l) was added to 175 l of the bacterial suspension and the reduction in absorbance at 450 nm was measured after 0 and 15 min at 22 C in a fluorimeter (BMG, Fluoro Star Galaxy). One unit of lysozyme activity was defined as a reduction in absorbance of 0·001 min 1. The units of lysozyme present in sera were obtained from a standard curve made with hen egg white lysozyme (Sigma).
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MYELOPEROXIDASE CONTENT
Total myeloperoxidase (MPO) content present in serum or inside leucocytes was measured according to Quade and Roth [21]. Fifteen l of serum were diluted with 135 l of HBSS without Ca +2 or Mg +2 in flat-bottomed 96 well plates. Then, 50 l of 20 mM 3,3 ,5,5 tetramethylbenzidine hydrochloride (TMB) (Sigma) and 5 mM H2O2 (both substrates of MPO and prepared daily) were added. The colour-change reaction was stopped after 2 min by adding 50 l of 4 M sulfuric acid (H2SO4). The optical density was read at 450 nm in a fluorimeter. Standard samples without serum were also analysed. To estimate the leucocyte MPO content, 106 head-kidney leucocytes per well were dispensed into flat-bottomed 96 well plates, washed and resuspended in 25 l of HBSS without Ca +2 or Mg +2. The leucocytes were then incubated for 15 min with 125 l of cetyltrimethylammonium bromide (CTAB) (Sigma) (0·02% in distilled water) and stirred at 40 rpm. Afterwards, the plates were centrifuged (400g, 10 min) and 150 l of the supernatants were transferred to a fresh 96 well plate, to which 25 l of 10 mM TMB and 5 mM H2O2 were added. After 2 min, 25 l of H2SO4 was added to stop the reaction and the absorbance was measured. Standard samples without leucocytes were also analysed. PHAGOCYTIC ACTIVITY
The phagocytic activity of gilthead seabream head-kidney leucocytes was studied by flow cytometry according to Esteban et al. [18]. Vibrio anguillarum strain R82 (serotype 01) was used as test particle and was grown and labelled with fluorescein isothiocyanate (FITC) (Sigma). FITC-labelled bacteria (10 l) were adjusted to 109 cells ml 1 in PBS, and added to each sample consisting of 50 l head-kidney leucocyte suspension previously adjusted to 107 cells ml 1 in sRPMI-1640. The samples were then centrifuged (400g, 5 min, 22 C), resuspended and incubated at 22 C for 30 min. At the end of the incubation time, the samples were placed on ice to stop phagocytosis and 400 l ice-cold PBS was added to each sample. The fluorescence of the extracellular bacteria (i.e. free bacteria and bacteria adhered to phagocytes but not ingested) was quenched by adding 40 l ice-cold trypan blue (0·4% in PBS) per sample. Standard samples of FITC-labelled V. anguillarum cells or head-kidney leucocytes were included in each phagocytosis assay. Samples incubated at 4 C were used as negative controls. All samples were analysed in a flow cytometer (Becton Dickinson) with an argon-ion laser adjusted to 488 nm. Analyses were performed on 3000 cells, which were acquired at a rate of 300 cells/s. Data were collected in the form of two-parameter side scatter (granularity) (SSC) and forward scatter (size) (FSC), and green fluorescence (FL1) and red fluorescence (FL2) dot plots or histograms were made on a computerised system. The fluorescence histograms represented the relative fluorescence on a logarithmic scale. The cytometer was set to analyse the phagocytic cells selected from all the leucocytes by their higher SSC and FSC parameters. Phagocytic ability was defined as the percentage of cells with one or more ingested bacteria (green-FITC fluorescent cells) within the phagocytic cell population. The relative number of ingested
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bacteria per cell (phagocytic capacity) was assessed by arbitrary units from the mean fluorescence intensity of the phagocytic cells. The quantitative study of the flow cytometric results was made using the statistical option of the Lysis Software Package (Becton Dickinson).
RESPIRATORY BURST ACTIVITY
The respiratory burst activity of gilthead seabream head-kidney leucocytes was studied by a chemiluminescence method [22]. Stock solutions of 1 mg ml 1 phorbol myristate acetate (PMA) (Sigma) in ethanol and 10 2 M luminol (Sigma) in dimethyl sulfoxide (DMSO) (Sigma) were prepared and stored at 20 C and 4 C, respectively. They were used to prepare the reactant solution containing final concentrations of 1 g PMA ml 1 and 10 4 M luminol in HBSS with calcium and magnesium. Leucocytes (100 l) and the solution containing PMA and luminol (100 l) were placed in the wells of a flatbottomed 96 well microtiter plate. The plate was shaken and immediately read in a chemiluminometer (BMG, Fluoro Star Galaxy). Measurement were performed in 30 cycles of 2 min each. The kinetics of the reactions were analysed and the maximum slope of each curve calculated. Backgrounds of luminiscence were calculated using reactant solutions containing luminol but not PMA. Controls contained only leucocytes.
FISH INTRAPERITONEALLY INJECTED WITH RETINOL ACETATE
Seventy specimens were placed in three laboratory aquaria and fed daily a commercial pellet diet at a ratio of 1% biomass. Specimens received an injection of 1 ml sterile phosphate bu#er (PBS) containing 0 (control), 0·05 or 0·30 mg retinol acetate 100 g 1 biomass. The accumulated mortality caused by retinol acetate was monitored daily for 30 days.
STATISTICAL ANALYSIS
The data in the figures are represented as means S.E. and were analysed by one-way analysis of variance (ANOVA) and the unpaired Student’s t-test. When the ANOVA test pointed to statistically significant (P
