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Changes in some innate defence parameters of seabream (Sparus aurata L.) induced by retinol acetate
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<0·05) di#erences between groups (control and retinol acetate), a comparison of means was applied.
III. Results GROWTH
Gilthead seabream growth was una#ected by dietary retinol acetate. The specific growth rate after 6 weeks of treatment ranged from 0·130·02 (control group) to 0·160·05 (highest retinol acetate dosage).
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Table 1. Serum lysozyme activity, expressed as iu ml 1, in seabream specimens fed retinol acetate supplemented diets. Data represent mean S.E. (n=6) Retinol acetate supplement (mg kg 1 diet) Control 50 150 300
Weeks of treatment 1
2
4
6
95·428·2 79·316·7 102·022·6 86·317·5
95·437·1 80·69·7 78·111·2 111·819·4
58·110·3 50·313·4 81·515·4 41·46·7
84·315·9 63·99·1 59·69·5 47·75·9
Table 2. Serum myeloperoxidase content, expressed as optical density at 450 nm, in seabream specimens fed retinol acetate supplemented diets. Data represent mean S.E. (n=6) Retinol acetate supplement (mg kg 1 diet) Control 50 150 300
Weeks of treatment 1
2
4
6
0·530·04 0·340·03 0·240·04 0·320·04
0·320·07 0·270·02 0·310·05 0·340·07
0·370·06 0·300·04 0·340·04 0·470·03
0·340·06 0·520·03 0·540·06 0·380·04
LYSOZYME ACTIVITY
The retinol acetate supplemented diets did not a#ect the serum lysozyme activity of gilthead seabream, which ranged from 41 to 115 iu ml 1 (Table 1). MYELOPEROXIDASE CONTENT
Gilthead seabream serum MPO content was not a#ected by the enriched retinol acetate diets (Table 2). The head-kidney leucocyte MPO content was increased to a statistically significant degree when gilthead seabream specimens were fed the highest retinol acetate supplemented diet for 2 and 4 weeks (P<0·05) (Fig. 1). Specimens fed 50 and 150 mg retinol acetate kg 1 supplemented diets also produced a statistically significant increase in the MPO content after 6 and 4 weeks of treatment (P<0·05), respectively. PHAGOCYTIC ACTIVITY
Head-kidney leucocytes isolated from retinol acetate fed seabream specimens showed slightly decreased phagocytic activity. Although there were only small di#erences, fish fed the 300 mg retinol acetate kg 1 diet for 2 weeks showed a statistically significant decrease in both phagocytic ability (% phagocytic cells) [Fig. 2(a)] and phagocytic capacity (mean fluorescence intensity per cell) [Fig. 2(b)] compared to the control group (non-supplemented diet).
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Fig. 1. Myeloperoxidase content, expressed as optical density at 450 nm, of gilthead seabream head-kidney leucocytes from specimens fed 0 ( ), 50 ( ), 150 ( ) or 300 ( ) mg retinol acetate-supplement kg 1 diet. Data represent the mean S.E. (n=6). Asterisks denote statistically significant di#erences (P<0·05) between control and retinol acetate fed groups. RESPIRATORY BURST
Head-kidney leucocytes from fish fed the highest retinol acetate supplement (300 mg kg 1) showed increased respiratory burst activity for all the assayed times although the increases were only statistically significant (P<0·05) after 1 and 2 weeks of treatment (Fig. 3). The retinol acetate supplement of 150 mg kg 1 also produced a statistically significant increase in the respiratory burst activity after 1 and 2 weeks of treatment. RETINOL ACETATE: INTRAPERITONEAL INJECTION
One day after injection, accumulated mortality was 28 and 48% for the lowest and highest retinol acetate dosages, respectively. This last figure increased slightly to 56% after the third day and remained constant until the end of the experiment in the group receiving the highest retinol acetate concentration. V. Discussion During recent decades many substances have been identified as enhancers of immune system activities [23, 24]. Several products from di#erent organisms, polymers, ions, vitamins and synthetic molecules have been identified as potent stimulants of the non-specific and/or specific immune system. In fish, some of them have also been identified as immunostimulants (e.g. -glucans, vitamins (C, E and A), yeasts and chitin). The term vitamin A normally includes all the compounds with the biological activity of retinol (retinols, retinyls, retinoic acid, carotenoids, etc.) [16, 25]. The most commonly used forms of vitamin A have been carotenoids (mainly -carotene), retinol acetate, retinyl palmitate and retinoic acid [5, 7, 26], which
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Fig. 2. Phagocytosis of gilthead seabream head-kidney leucocytes from specimens fed 0 ( ), 50 ( ), 150 ( ) or 300 ( ) mg retinol acetate-supplement kg 1 diet. (a) Phagocytic ability expressed as the percentage of phagocytic cells. (b) Phagocytic capacity expressed as the mean fluorescence intensity cell 1 in arbitrary units. Data represent the mean S.E. (n=6). Asterisk denotes statistically significant di#erences (P<0·05) between control and retinol acetate fed groups.
are more stable and more easily handled than retinol. In mammals, vitamin A is widely known as an anti-infective vitamin [27] and has been shown to enhance antibody production, proliferative responses of T and B lymphocytes to mitogens, cytotoxic activity (mediated by T lymphocytes, natural killer cells or macrophages), phagocytosis and the secretion of tumour necrosis factor-; it also provokes a decrease in tumour burden [5–7, 11, 27]. In spite of all these observed e#ects it is still not clearly understood how vitamin A works on the immune system. Several authors have pointed out that perhaps its e#ects are due to its antioxidant activity, its adjuvant properties or more generally, its involvement in some biosynthetic pathways [1, 25]. The most widely studied vitamins with immunostimulant properties in fish are vitamins C and E [28–35], and although the impact of vitamin A and related compounds on the mammalian immune system functions has been described there are a few papers concerning vitamin A and its role on the immune system in fish [14–17]. Moreover, the available data often seem controversial and do not clarify the vitamin’s role in the teleost fish immune system.
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Fig. 3. Respiratory burst activity of gilthead seabream head-kidney leucocytes from specimens fed 0 ( ), 50 ( ), 150 ( ) or 300 ( ) mg retinol acetate-supplement kg 1 diet. Data represent the mean S.E. (n=6). Asterisks denote statistically significant di#erences (P<0·05) between control and retinol acetate fed groups.
It has been shown for several immunostimulants that di#erent administration ways have di#erent e#ects on the fish immune system, even for the same substance tested. Although intraperitoneal injection has been seen to be the most rapid and e#ective administration way the incorporation in the diet is regarded as the most suitable for fish farming [23, 36, 37]. For this reason both administration ways were studied in the present work. High vitamin A dietary intake is shown to act as an immunostimulant for fish-farming purposes in gilthead seabream. Specimens fed dietary supplements of retinol acetate did not show any external signs of toxicity, as has been described for some mammals and fish species after hypo- or hypervitaminosis [12, 38], and no di#erences in the specific growth rate were observed after retinol acetate treatment. As regards the innate humoral immune system of the seabream specimens, neither lysozyme activity nor myeloperoxidase content in serum was a#ected by the use of vitamin A-supplemented diets. In a previous study in Atlantic salmon, humoral response (represented as serum antibody levels and bactericidal activity to Aeromonas salmonicida and antiprotease activity) was increased by the high retinol acetate-supplemented diets [16]. Rainbow trout specimens receiving -carotene supplemented diets for 12 weeks increased their serum immunoglogulin levels and complement activity [17]. In mammals, vitamin A has been shown to be involved in the biosynthesis of antiproteases [39] and lysozyme [40]. Among the humoral defence responses antibody production has been the most enhanced factor by vitamin A status [1], perhaps due to its adjuvant properties [41]. However, little and controversial information exist regarding this humoral response in fish. Innate cellular defence responses including respiratory burst activity and leucocyte myeloperoxidase content, but not phagocytosis, were enhanced by dietary retinol acetate intake. However, the highest assayed concentration of retinol acetate slightly decreased the phagocytic ability and capacity of fish
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after 2 weeks of treatment. Treatment with retinol acetate over a period of time not comparable with that studied in the present work (4 months) produced no e#ect on the phagocytic responses of Atlantic salmon [16] while rainbow trout increased their phagocytic activity after a 400 mg -carotene kg 1 diet for 12 weeks [17]. Di#erent results have been observed in mammals. For example, retinyl palmitate included in the diet for 7 weeks increased phagocytosis, tumoricidal activity and IL-1 production in peritoneal exudate macrophages of mice [7]. Calf leucocytes incubated in vitro with retinyl palmitate showed increased neutrophil phagocytosis although bactericidal activity was slightly decreased in the case of neutrophils and macrophages [42]. Although only a few studies have been carried out to investigate the e#ect of vitamin A on fish macrophage functions, to date none has described a positive role in this respect. Perhaps the di#erences observed between fish and mammals are due to dosage protocols or experimental times or even, to the use of di#erent vitamin A sources (retinol acetate or retinyl palmitate). Both gilthead seabream head-kidney leucocyte respiratory burst activity and leucocyte myeloperoxidase (MPO) content were enhanced after retinol acetate treatment. In the phenomenon known as respiratory or oxidative burst, large amounts of reactive oxygen intermediates (ROIs) are produced [43]. In this work the superoxide radical (O2 ) which is indicative of respiratory burst activity was measured by chemiluminescence. The MPO enzyme is also involved in that enzymatic cascade. For this reason, data regarding the e#ect of a particular treatment on respiratory burst activity and MPO content in leucocytes should be in agreement because they are di#erent measures of the same enzymatic cascade. In this study, both were stimulated by dietary retinol acetate intake, although di#erences were observed in the vitamin concentration and administration time at which the maximum values were attained. This could have been due to di#erences in the sensitivity of the methods used to measure each parameter. The present results are in agreement with the antioxidant properties assigned to vitamin A. We performed an experiment in which retinol acetate was administered by i.p. injection in order to compare the e#ects on the seabream innate defence system of the same substance administered in di#erent ways. For this, the same amount of retinol acetate that was included in the diet per day was prepared and fish were given a single injection. A 100 g specimen fed the lowest or the highest retinol acetate supplemented diet (50 or 300 mg kg 1 diet) received 1 g of diet daily, which is equivalent to 0·05 or 0·3 mg retinol acetate 100 g 1 biomass, respectively. Three groups of specimens were injected with 1 ml of PBS containing 0 (control), 0·05 or 0·3 mg retinol acetate 100 g 1 biomass. Unexpectedly, the amounts of retinol acetate injected were highly toxic for seabream specimens. One day after injection, 28 and 48% of specimens were found dead in the groups receiving 0·05 and 0·3 retinol acetate 100 g 1 biomass, respectively. From that point onwards, only the group injected with the highest retinol acetate concentration increased the accumulated mortality to 56% (on day 3). The results indicate the toxicity of high amounts of retinol acetate when administered by intraperitoneal injection, compared to the dietary administration of the same daily amount for a long period of time (up to 6 weeks). A single study carried out in channel catfish
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involving retinol acetate administration resulted in increased leucocyte tumoricidal activity but the intraperitoneal injection was more rapid and e#ective than the dietary supplement intake [15]. In the above study, the vitamin A supplements were up to five times the normal vitamin requirement, which are not comparable to the supplements assayed in this work. For other substances, such as chitin, injection was also more rapid and e#ective than oral administration [37, 44]. To conclude, seabream specimens fed retinol acetate supplemented diets showed enhanced respiratory burst activity and increased leucocyte MPO content, while humoral factors were una#ected. However, equivalent amounts of retinol acetate were toxic, even lethal, when injected intraperitoneally. The present results are in accordance with the antioxidant properties suggested for vitamin A. Important and significant di#erences exist between the present results and those obtained in previous works using other fish species. More studies should be carried out to clarify the role played by vitamin A in the fish immune system. References 1 Ross, A. C. (1992). Vitamin A status: Relationship to immunity and the antibody response. Experimental Biology & Medicine 200, 303–320. 2 Underwood, B. A. (1984). Vitamin A in animal and human nutrition. In The Retinoids (M. B. Sporn, A. B. Roberts & D. S. Goodman, eds) pp. 281–392. Orlando, FL: Academic Press. 3 Bendich, A. & Olson, J. A. (1989). Biological actions of carotenoids. The FASEB Journal 3, 1927–1932. 4 McMichael, H. (1965). Inhibition of the growth of Shope rabbit papilloma by hypervitaminosis A. Cancer Research 25, 947–955. 5 Dennert, G., Crowley, C., Kouba, J. & Lotan, R. (1979). Retinoic acid stimulation of the induction of mouse killer T-cells in allogeneic and syngeneic systems. Journal National Cancer Institute 62, 89–94. 6 Goldfarb, R. H. & Herberman, R. B. (1981). Natural killer cell reactivity: regulatory interactions among phorbol ester, interferon, cholera toxin and retinoic acid. Journal of Immunology 126, 2129–2135. 7 Moriguchi, S., Werner, L. & Watson, R. R. (1985). High dietary vitamin A (retinyl palmitate) and cellular immune functions in mice. Immunology 56, 169–177. 8 Lessard, M., Hutchings, D. & Cave, N. A. (1997). Cell-mediated and humoral immune responses in broiler chickens maintained on diets containing di#erent levels of vitamin A. Poultry Science 76, 1368–1378. 9 Kashida, T., Narasaki, N., Yano, T., Tsuzurahara, K. & Takeyama, S. (1998). Augmentation of tumor immunity in mice by intralesional injection of vitamin A. Biology and Pharmacology Bulletin 21, 339–345. 10 Kim, H. W., Chew, B. P., Wong, T. S., Park, J. S., Weng, B. B., Byrne, K. M., Hayek, M. G. & Reinhart, G. A. (2000). Modulation of humoral and cell-mediated immune responses by dietary lutein in cats. Veterinary Immunology and Immunopathology 73, 331–341. 11 Jurin, M. & Tannock, I. F. (1972). Influence of vitamin A on immunological response. Immunology 23, 283–287. 12 Clive, E., West, C. E., Rombout, J. H. W. M., Van der Zijpp, A. P. & Sijtsma, S. R. (1991). Vitamin A and immune function. Proceedings of the Nutrition Society 50, 251–262. 13 Schiedt, K., Leuenberger, F. J., Vecchi, M. & Glinz, E. (1985). Absorption, retention and metabolic transformations of carotenoids in rainbow trout, salmon and chicken. Pure and Applied Chemistry 57, 685–692.
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14 Malikova, E. M., Apine, S. O. & Shaldaeva, R. E. (1961). Use of vitamins as prophylactic and therapeutic measures in immature Salmo salar in fish hatcheries. Tr. Nauchn. Inst. Rybn. Khoz. Latviisk. SSR. 3, 445–452. 15 Evans, D. L., Graves, S. S., Blazer, V. S., Dawe, D. L. & Graztek, J. B. (1984). Immunoregulation of fish non-specific cytotoxic cell activity by retinol acetate but not poly I:C. Comparative Immunology and Microbiology Infection Diseases 7, 91–100. 16 Thompson, I., Fletcher, T. C., Houlihan, D. F. & Secombes, C. J. (1994). The e#ect of dietary vitamin A on the immunocompetence of Atlantic salmon (Salmo salar L.). Fish Physiology and Biochemistry 12, 513–523. 17 Amar, E. C., Kiron, V., Satoh, S., Okamoto, N. & Watanabe, T. (2000). E#ects of dietary -carotene on the immune response of rainbow trout Oncorhynchus mykiss. Fisheries Science 66, 1068–1075. 18 Esteban, M. A., Mulero, V., Mun˜oz, J. & Meseguer, J. (1998). Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy. Cell & Tissue Research 293, 133–141. 19 Ricker, W. E. (1979). Growth rates and models. In Fish Physiology (W. S. Hoar, P. J. Randal & J. R. Brett, eds) pp. 677–743. New York: Academic Press. 20 Parry, R. M., Chandau, R. C. & Shahani, R. M. (1965). A rapid and sensitive assay of muramidase. Proceedings of the Society of Experimental Biology and Medicine 119, 384–386. 21 Quade, M. J. & Roth, J. A. (1997). A rapid, direct assay to measure degranulation of bovine neutrophil primary granules. Veterinary Immunology and Immunopathology 58, 239–248. 22 Bayne, C. J. & Levy, S. (1991). Modulation of the oxidative burst in trout myeloid cells by adrenocorticotropic hormone and catecholamines: mechanisms of action. Journal of Leukocyte Biology 50, 554–560. 23 Blazer, V. S. (1992). Nutrition and disease resistance in fish. Annual Review of Fish Diseases 2, 309–323. 24 Sakai, M. (1999). Current research status of fish immunostimulants. Aquaculture 172, 63–92. 25 Bendich, A. (1992). Physiological role of antioxidants in the immune system. Journal of Dairy Science 76, 2789–2794. 26 Bendich, A. (1991). Beta-carotene and the immune response. Proceedings of the Nutrition Society 50, 263–274. 27 Green, H. N. & Mellanby, E. (1928). Vitamin A as an anti-infective agent. British Medical Journal 2, 691–696. 28 Blazer, V. S. & Wolke, R. E. (1984). The e#ects of -tocopherol on the immune response and non-specific resistance factors of rainbow trout (Salmo gairdneri Richardson). Aquaculture 37, 1–9. 29 Hardie, L. J., Fletcher, T. C. & Secombes, C. J. (1990). The e#ect of vitamin E on the immune response of the Atlantic salmon (Salmo salar L.). Aquaculture 87, 1–13. 30 Verlhac, V., N’doye, A., Gabaudan, J., Trotaud, D. & Deschaux, P. (1993). Vitamin nutrition and fish immunity: Influence of antioxidant vitamins (C and E) on immune responses of rainbow trout. In Fish Nutrition in Practice (S. J. Kaushik & P. Luquet, eds) pp. 167–177. Paris: INRA. 31 Ortun˜o, J., Esteban, M. A. & Meseguer, J. (1999). E#ect of high dietary intake of vitamin C on non-specific immune response of gilthead seabream (Sparus aurata L.). Fish & Shellfish Immunology 9, 429–443. 32 Ortun˜o, J., Esteban, M. A. & Meseguer, J. (2000). High dietary intake of -tocopherol acetate enhances the non-specific immune response of gilthead seabream (Sparus aurata L.). Fish & Shellfish Immunology 10, 293–307. 33 Ortun˜o, J., Cuesta, A., Esteban, M. A. & Meseguer, J. (2001). E#ect of oral administration of high vitamins C and E dosages on the gilthead seabream (Sparus aurata L.) innate immune system. Veterinary Immunology and Immunopathology 79, 167–180.
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34 Cuesta, A., Esteban, M. A., Ortun˜o, J. & Meseguer, J. (2001). Vitamin E increases natural cytotoxic activity in seabream (Sparus aurata L.). Fish & Shellfish Immunology 11, 293–302. 35 Cuesta, A., Esteban, M. A. & Meseguer, J. (in press). Natural cytotoxic activity in seabream (Sparus aurata L.) and its modulation by vitamin C. Fish & Shellfish Immunology doi:10.1006/fsim.2001.0359. 36 Anderson, D. P. (1992). Immunostimulants, adjuvants, and vaccine carriers in fish: applications to aquaculture. Annual Review of Fish Diseases 1992, 281–307. 37 Esteban, M. A., Cuesta, A., Ortun˜o, J. & Meseguer, J. (2001). Immunomodulatory e#ects of dietary intake of chitin on gilthead seabream (Sparus aurata L.) innate immune response. Fish & Shellfish Immunology 11, 303–315. 38 Halver, J. E. (1972). The vitamins. In Fish Nutrition (J. E. Halver, eds) pp. 29–123. New York/London: Academic Press. 39 Kiorpes, T. C., Salvatore, J. M. & Wolf, G. (1976). A plasma glycoprotein depressed by vitamin A deficiency in the rat: 1-macroglobulin. Journal of Nutrition 106, 1659–1667. 40 Mohanram, M., Reddy, V. & Mishra, S. (1974). Lysozyme activity in plasma and leucocytes in malnourished children. British Medical Journal 32, 313–316. 41 Dresser, D. W. (1968). Adjuvanticity of vitamin A. Nature 217, 527–529. 42 Eicher, S. D., Morril, J. L. & Blecha, F. (1993). Vitamin concentration and function of leukocytes from dairy calves supplemented with vitamin A, vitamin E, and -carotene in vitro. Journal of Dairy Science 77, 560–565. 43 Ortun˜o, J., Esteban, M. A. & Meseguer, J. (2000). Kinetics of hydrogen peroxide production during in vitro respiratory burst of seabream (Sparus aurata L.) head-kidney leucocytes, as measured by a flow cytometric method. Fish & Shellfish Immunology 10, 725–729. 44 Esteban, M. A., Mulero, V., Cuesta, A., Ortun˜o, J. & Meseguer, J. (2000). E#ects of injecting chitin particles on the innate immune response of gilthead seabream (Sparus aurata L.). Fish & Shellfish Immunology 10, 543–554.
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<0·05) di#erences between groups (control and retinol acetate), a comparison of means was applied.
III. Results GROWTH
Gilthead seabream growth was una#ected by dietary retinol acetate. The specific growth rate after 6 weeks of treatment ranged from 0·130·02 (control group) to 0·160·05 (highest retinol acetate dosage).
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Table 1. Serum lysozyme activity, expressed as iu ml 1, in seabream specimens fed retinol acetate supplemented diets. Data represent mean S.E. (n=6) Retinol acetate supplement (mg kg 1 diet) Control 50 150 300
Weeks of treatment 1
2
4
6
95·428·2 79·316·7 102·022·6 86·317·5
95·437·1 80·69·7 78·111·2 111·819·4
58·110·3 50·313·4 81·515·4 41·46·7
84·315·9 63·99·1 59·69·5 47·75·9
Table 2. Serum myeloperoxidase content, expressed as optical density at 450 nm, in seabream specimens fed retinol acetate supplemented diets. Data represent mean S.E. (n=6) Retinol acetate supplement (mg kg 1 diet) Control 50 150 300
Weeks of treatment 1
2
4
6
0·530·04 0·340·03 0·240·04 0·320·04
0·320·07 0·270·02 0·310·05 0·340·07
0·370·06 0·300·04 0·340·04 0·470·03
0·340·06 0·520·03 0·540·06 0·380·04
LYSOZYME ACTIVITY
The retinol acetate supplemented diets did not a#ect the serum lysozyme activity of gilthead seabream, which ranged from 41 to 115 iu ml 1 (Table 1). MYELOPEROXIDASE CONTENT
Gilthead seabream serum MPO content was not a#ected by the enriched retinol acetate diets (Table 2). The head-kidney leucocyte MPO content was increased to a statistically significant degree when gilthead seabream specimens were fed the highest retinol acetate supplemented diet for 2 and 4 weeks (P<0·05) (Fig. 1). Specimens fed 50 and 150 mg retinol acetate kg 1 supplemented diets also produced a statistically significant increase in the MPO content after 6 and 4 weeks of treatment (P<0·05), respectively. PHAGOCYTIC ACTIVITY
Head-kidney leucocytes isolated from retinol acetate fed seabream specimens showed slightly decreased phagocytic activity. Although there were only small di#erences, fish fed the 300 mg retinol acetate kg 1 diet for 2 weeks showed a statistically significant decrease in both phagocytic ability (% phagocytic cells) [Fig. 2(a)] and phagocytic capacity (mean fluorescence intensity per cell) [Fig. 2(b)] compared to the control group (non-supplemented diet).
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Fig. 1. Myeloperoxidase content, expressed as optical density at 450 nm, of gilthead seabream head-kidney leucocytes from specimens fed 0 ( ), 50 ( ), 150 ( ) or 300 ( ) mg retinol acetate-supplement kg 1 diet. Data represent the mean S.E. (n=6). Asterisks denote statistically significant di#erences (P<0·05) between control and retinol acetate fed groups. RESPIRATORY BURST
Head-kidney leucocytes from fish fed the highest retinol acetate supplement (300 mg kg 1) showed increased respiratory burst activity for all the assayed times although the increases were only statistically significant (P<0·05) after 1 and 2 weeks of treatment (Fig. 3). The retinol acetate supplement of 150 mg kg 1 also produced a statistically significant increase in the respiratory burst activity after 1 and 2 weeks of treatment. RETINOL ACETATE: INTRAPERITONEAL INJECTION
One day after injection, accumulated mortality was 28 and 48% for the lowest and highest retinol acetate dosages, respectively. This last figure increased slightly to 56% after the third day and remained constant until the end of the experiment in the group receiving the highest retinol acetate concentration. V. Discussion During recent decades many substances have been identified as enhancers of immune system activities [23, 24]. Several products from di#erent organisms, polymers, ions, vitamins and synthetic molecules have been identified as potent stimulants of the non-specific and/or specific immune system. In fish, some of them have also been identified as immunostimulants (e.g. -glucans, vitamins (C, E and A), yeasts and chitin). The term vitamin A normally includes all the compounds with the biological activity of retinol (retinols, retinyls, retinoic acid, carotenoids, etc.) [16, 25]. The most commonly used forms of vitamin A have been carotenoids (mainly -carotene), retinol acetate, retinyl palmitate and retinoic acid [5, 7, 26], which
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Fig. 2. Phagocytosis of gilthead seabream head-kidney leucocytes from specimens fed 0 ( ), 50 ( ), 150 ( ) or 300 ( ) mg retinol acetate-supplement kg 1 diet. (a) Phagocytic ability expressed as the percentage of phagocytic cells. (b) Phagocytic capacity expressed as the mean fluorescence intensity cell 1 in arbitrary units. Data represent the mean S.E. (n=6). Asterisk denotes statistically significant di#erences (P<0·05) between control and retinol acetate fed groups.
are more stable and more easily handled than retinol. In mammals, vitamin A is widely known as an anti-infective vitamin [27] and has been shown to enhance antibody production, proliferative responses of T and B lymphocytes to mitogens, cytotoxic activity (mediated by T lymphocytes, natural killer cells or macrophages), phagocytosis and the secretion of tumour necrosis factor-; it also provokes a decrease in tumour burden [5–7, 11, 27]. In spite of all these observed e#ects it is still not clearly understood how vitamin A works on the immune system. Several authors have pointed out that perhaps its e#ects are due to its antioxidant activity, its adjuvant properties or more generally, its involvement in some biosynthetic pathways [1, 25]. The most widely studied vitamins with immunostimulant properties in fish are vitamins C and E [28–35], and although the impact of vitamin A and related compounds on the mammalian immune system functions has been described there are a few papers concerning vitamin A and its role on the immune system in fish [14–17]. Moreover, the available data often seem controversial and do not clarify the vitamin’s role in the teleost fish immune system.
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Fig. 3. Respiratory burst activity of gilthead seabream head-kidney leucocytes from specimens fed 0 ( ), 50 ( ), 150 ( ) or 300 ( ) mg retinol acetate-supplement kg 1 diet. Data represent the mean S.E. (n=6). Asterisks denote statistically significant di#erences (P<0·05) between control and retinol acetate fed groups.
It has been shown for several immunostimulants that di#erent administration ways have di#erent e#ects on the fish immune system, even for the same substance tested. Although intraperitoneal injection has been seen to be the most rapid and e#ective administration way the incorporation in the diet is regarded as the most suitable for fish farming [23, 36, 37]. For this reason both administration ways were studied in the present work. High vitamin A dietary intake is shown to act as an immunostimulant for fish-farming purposes in gilthead seabream. Specimens fed dietary supplements of retinol acetate did not show any external signs of toxicity, as has been described for some mammals and fish species after hypo- or hypervitaminosis [12, 38], and no di#erences in the specific growth rate were observed after retinol acetate treatment. As regards the innate humoral immune system of the seabream specimens, neither lysozyme activity nor myeloperoxidase content in serum was a#ected by the use of vitamin A-supplemented diets. In a previous study in Atlantic salmon, humoral response (represented as serum antibody levels and bactericidal activity to Aeromonas salmonicida and antiprotease activity) was increased by the high retinol acetate-supplemented diets [16]. Rainbow trout specimens receiving -carotene supplemented diets for 12 weeks increased their serum immunoglogulin levels and complement activity [17]. In mammals, vitamin A has been shown to be involved in the biosynthesis of antiproteases [39] and lysozyme [40]. Among the humoral defence responses antibody production has been the most enhanced factor by vitamin A status [1], perhaps due to its adjuvant properties [41]. However, little and controversial information exist regarding this humoral response in fish. Innate cellular defence responses including respiratory burst activity and leucocyte myeloperoxidase content, but not phagocytosis, were enhanced by dietary retinol acetate intake. However, the highest assayed concentration of retinol acetate slightly decreased the phagocytic ability and capacity of fish
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after 2 weeks of treatment. Treatment with retinol acetate over a period of time not comparable with that studied in the present work (4 months) produced no e#ect on the phagocytic responses of Atlantic salmon [16] while rainbow trout increased their phagocytic activity after a 400 mg -carotene kg 1 diet for 12 weeks [17]. Di#erent results have been observed in mammals. For example, retinyl palmitate included in the diet for 7 weeks increased phagocytosis, tumoricidal activity and IL-1 production in peritoneal exudate macrophages of mice [7]. Calf leucocytes incubated in vitro with retinyl palmitate showed increased neutrophil phagocytosis although bactericidal activity was slightly decreased in the case of neutrophils and macrophages [42]. Although only a few studies have been carried out to investigate the e#ect of vitamin A on fish macrophage functions, to date none has described a positive role in this respect. Perhaps the di#erences observed between fish and mammals are due to dosage protocols or experimental times or even, to the use of di#erent vitamin A sources (retinol acetate or retinyl palmitate). Both gilthead seabream head-kidney leucocyte respiratory burst activity and leucocyte myeloperoxidase (MPO) content were enhanced after retinol acetate treatment. In the phenomenon known as respiratory or oxidative burst, large amounts of reactive oxygen intermediates (ROIs) are produced [43]. In this work the superoxide radical (O2 ) which is indicative of respiratory burst activity was measured by chemiluminescence. The MPO enzyme is also involved in that enzymatic cascade. For this reason, data regarding the e#ect of a particular treatment on respiratory burst activity and MPO content in leucocytes should be in agreement because they are di#erent measures of the same enzymatic cascade. In this study, both were stimulated by dietary retinol acetate intake, although di#erences were observed in the vitamin concentration and administration time at which the maximum values were attained. This could have been due to di#erences in the sensitivity of the methods used to measure each parameter. The present results are in agreement with the antioxidant properties assigned to vitamin A. We performed an experiment in which retinol acetate was administered by i.p. injection in order to compare the e#ects on the seabream innate defence system of the same substance administered in di#erent ways. For this, the same amount of retinol acetate that was included in the diet per day was prepared and fish were given a single injection. A 100 g specimen fed the lowest or the highest retinol acetate supplemented diet (50 or 300 mg kg 1 diet) received 1 g of diet daily, which is equivalent to 0·05 or 0·3 mg retinol acetate 100 g 1 biomass, respectively. Three groups of specimens were injected with 1 ml of PBS containing 0 (control), 0·05 or 0·3 mg retinol acetate 100 g 1 biomass. Unexpectedly, the amounts of retinol acetate injected were highly toxic for seabream specimens. One day after injection, 28 and 48% of specimens were found dead in the groups receiving 0·05 and 0·3 retinol acetate 100 g 1 biomass, respectively. From that point onwards, only the group injected with the highest retinol acetate concentration increased the accumulated mortality to 56% (on day 3). The results indicate the toxicity of high amounts of retinol acetate when administered by intraperitoneal injection, compared to the dietary administration of the same daily amount for a long period of time (up to 6 weeks). A single study carried out in channel catfish
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involving retinol acetate administration resulted in increased leucocyte tumoricidal activity but the intraperitoneal injection was more rapid and e#ective than the dietary supplement intake [15]. In the above study, the vitamin A supplements were up to five times the normal vitamin requirement, which are not comparable to the supplements assayed in this work. For other substances, such as chitin, injection was also more rapid and e#ective than oral administration [37, 44]. To conclude, seabream specimens fed retinol acetate supplemented diets showed enhanced respiratory burst activity and increased leucocyte MPO content, while humoral factors were una#ected. However, equivalent amounts of retinol acetate were toxic, even lethal, when injected intraperitoneally. The present results are in accordance with the antioxidant properties suggested for vitamin A. Important and significant di#erences exist between the present results and those obtained in previous works using other fish species. More studies should be carried out to clarify the role played by vitamin A in the fish immune system. References 1 Ross, A. C. (1992). Vitamin A status: Relationship to immunity and the antibody response. Experimental Biology & Medicine 200, 303–320. 2 Underwood, B. A. (1984). Vitamin A in animal and human nutrition. In The Retinoids (M. B. Sporn, A. B. Roberts & D. S. Goodman, eds) pp. 281–392. Orlando, FL: Academic Press. 3 Bendich, A. & Olson, J. A. (1989). Biological actions of carotenoids. The FASEB Journal 3, 1927–1932. 4 McMichael, H. (1965). Inhibition of the growth of Shope rabbit papilloma by hypervitaminosis A. Cancer Research 25, 947–955. 5 Dennert, G., Crowley, C., Kouba, J. & Lotan, R. (1979). Retinoic acid stimulation of the induction of mouse killer T-cells in allogeneic and syngeneic systems. Journal National Cancer Institute 62, 89–94. 6 Goldfarb, R. H. & Herberman, R. B. (1981). Natural killer cell reactivity: regulatory interactions among phorbol ester, interferon, cholera toxin and retinoic acid. Journal of Immunology 126, 2129–2135. 7 Moriguchi, S., Werner, L. & Watson, R. R. (1985). High dietary vitamin A (retinyl palmitate) and cellular immune functions in mice. Immunology 56, 169–177. 8 Lessard, M., Hutchings, D. & Cave, N. A. (1997). Cell-mediated and humoral immune responses in broiler chickens maintained on diets containing di#erent levels of vitamin A. Poultry Science 76, 1368–1378. 9 Kashida, T., Narasaki, N., Yano, T., Tsuzurahara, K. & Takeyama, S. (1998). Augmentation of tumor immunity in mice by intralesional injection of vitamin A. Biology and Pharmacology Bulletin 21, 339–345. 10 Kim, H. W., Chew, B. P., Wong, T. S., Park, J. S., Weng, B. B., Byrne, K. M., Hayek, M. G. & Reinhart, G. A. (2000). Modulation of humoral and cell-mediated immune responses by dietary lutein in cats. Veterinary Immunology and Immunopathology 73, 331–341. 11 Jurin, M. & Tannock, I. F. (1972). Influence of vitamin A on immunological response. Immunology 23, 283–287. 12 Clive, E., West, C. E., Rombout, J. H. W. M., Van der Zijpp, A. P. & Sijtsma, S. R. (1991). Vitamin A and immune function. Proceedings of the Nutrition Society 50, 251–262. 13 Schiedt, K., Leuenberger, F. J., Vecchi, M. & Glinz, E. (1985). Absorption, retention and metabolic transformations of carotenoids in rainbow trout, salmon and chicken. Pure and Applied Chemistry 57, 685–692.
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