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Vitamin E increases natural cytotoxic activity in seabream (Sparus aurata L.)
Fish & Shellfish Immunology (2001) 11, 293–302 doi:10.1006/fsim.2000.0316 Available online at http://www.idealibrary.com on
Vitamin E increases natural cytotoxic activity in seabream (Sparus aurata L.) A. CUESTA, M. A. ESTEBAN, J. ORTUÑO AND J. MESEGUER* Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain (Received 27 March 2000, accepted after revision 9 October 2000) The natural cytotoxic activity of head-kidney leucocytes from gilthead seabream (Sparus aurata L.), after in vitro and in vivo vitamin E treatment, against tumor cells was studied by flow cytometry. Leucocytes were incubated in culture medium with di#erent vitamin E supplementations (0·01–10 g ml 1) for 6, 24 or 48 h and the results demonstrate that all the assayed vitamin E supplementations significantly enhanced the natural cytotoxic activity of leucocytes. To determine the e#ect of a high dietary level of vitamin E on this activity, fish were fed with 0 (control), 600, 1200 or 1800 mg of vitamin E supplementation kg 1 diet for 2, 4 or 6 weeks. After 2 and 4 weeks of treatment, the natural cytotoxic activity was significantly enhanced at the highest (1·8 g kg 1 diet) and lowest (600 mg kg 1 diet) vitamin E supplement dosage, respectively. No e#ect of the vitamin E supplemented diet on seabream leucocyte natural cytotoxic cell activity was observed after 6 weeks of treatment. 2001 Academic Press Key words:
vitamin E, natural cytotoxic activity, non-specific cytotoxic cells (NCC), leucocytes, gilthead seabream (Sparus aurata L.).
I. Introduction Vitamin E, besides being a very important lipid-soluble micronutrient (Tu et al., 1995), is a normal constituent of all cellular membranes, being particularly abundant in immune cell membranes, where it is an essential component for cell functioning. Furthermore, due to its antioxidant properties, vitamin E interacts with other nutrients, especially polyunsaturated fatty acids (Beharka et al., 1997). The role played by vitamin E in several immunological responses has been studied in mammals (Meydani et al., 1986; Moriguchi et al., 1990; Ndiweni & Finch, 1995; Beharka et al., 1997) and teleosts (Cowey et al., 1981; Hung et al., 1982; Blazer & Wolke, 1984; Hardie et al., 1990; Mulero et al., 1998; Ortun˜o et al., 2000). Mammals fed vitamin E-depleted diets showed depressed immune activities such as resistance to infection, specific antibody responses, splenic antibody forming cells, in vitro mitogenic responses of lymphocytes, reticuloendothelial system clearance or phagocytic index. All these immune functions were enhanced as a consequence of -tocopherol intake (Meydani & *Corresponding author. E-mail: [email protected] 1050–4648/01/040293+10 $35.00/0
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Blumberg, 1992). The available data on the e#ect of vitamin E on the fish immune system are particularly scarce although some similarities with mammals may be observed. For example, humoral immune responses, such as complement activity and haemagglutination and haemolysin titres, were depressed in trout fed vitamin-E depleted diets (Blazer & Wolke, 1984), while Atlantic salmon fed a vitamin E-supplemented diet showed una#ected humoral responses (Hardie et al., 1990). Similarly, T and B cell responses and phagocytic activity were depressed in trout fed vitamin E-depleted diets (Blazer & Wolke, 1984), while Atlantic salmon fed vitamin E-supplemented diet exhibited increased phagocytic activity although neither their respiratory burst activity or their lymphokine production were a#ected (Hardie et al., 1990). Moreover, in our laboratory we have previously described both the in vitro and in vivo e#ect of vitamin E (-tocopherol) on the gilthead seabream non-specific immune response, particularly with respect to leucocyte migration and phagocytic and respiratory burst activities (Mulero et al., 1998; Ortun˜ o et al., 2000). Although natural cytotoxic cell activity is a major immune defence mechanism against virus-infected cells, tumor cells and parasites, no data exist about the e#ect of vitamin E on the fish cytotoxic cell response. The aim of the present work was to study the in vitro and in vivo e#ect of vitamin E on gilthead seabream leucocyte natural cytotoxic activity. II. Materials and Methods ANIMALS
One hundred 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 a running seawater aquaria (flow rate 1500 l h 1) at 20 C with a natural photoperiod. Animals were fed a commercial pellet diet (ProAqua S.A., Palencia, Spain) at a rate of 1·5% body weight day 1.
HEAD-KIDNEY LEUCOCYTE ISOLATION
The animals were anaesthetized with 100 mg l 1 MS222 (Sandoz) and the head-kidney leucocytes were isolated under sterile conditions. Briefly, the 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 nylon mesh (mesh size 102 m). Head-kidney cell suspensions were layered over a 48% Percoll density gradient (Pharmacia) and centrifuged at 400g for 30 min at 4 C (Esteban et al., 1998). After centrifugation, the band of leucocytes above the 48% interface were collected with a Pasteur pipette and washed twice. Cell
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viability was higher than 98%, as determined by the trypan blue exclusion test. VITAMIN E SUPPLEMENTED CULTURE MEDIUM
Vitamin E was prepared by dissolving -tocopherol acetate (Sigma) in acetone (10 mg ml 1). This solution was diluted in sRPMI culture medium to obtain a 0·1 mg ml 1 vitamin E stock solution, which was aliquoted and stored at 80 C until use. To study the in vitro e#ect of vitamin E on the head-kidney leucocyte natural cytotoxic activity, previously isolated leucocytes were adjusted to 5·55106 cells ml 1 in sRPMI-1640 medium supplemented with 10% fetal calf serum (FCS). Then, 450 l of the leucocyte suspensions were dispensed into the wells of a flat bottomed 48 well plate (Nunc). Fifty l of the vitamin E stock solution or 10 times serially diluted stock solution were added to each well, to give a final vitamin E concentration ranging from 0·01–10 g ml 1. Fifty l of culture medium were used for the controls. The samples were incubated at 22 C for 6, 24 or 48 h. The viability of leucocytes incubated with acetone or di#erent vitamin E-supplemented culture medium was also monitored by flow cytometry. VITAMIN E SUPPLEMENTED DIETS
Four experimental diets were prepared in the laboratory from a commercial pellet diet (-tocopherol content 100 mg kg 1). For this, three vitamin E solutions of 24, 48 and 72 mg -tocopherol acetate (Sigma) ml 1 fish oil were made. 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 600, 1200 and 1800 mg vitamin E kg 1 diet. The nonsupplemented diet (control) was prepared by spraying the feed with fish oil alone. The -tocopherol level of the fish oil used to dilute the vitamin E was less than 1 g ml 1, as determined by high-pressure-liquid-chromatography (HPLC) (Ortun˜ o et al., 2000). FISH: TREATMENT AND SAMPLING
The fish were divided randomly into four groups, which were distributed in 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%) each day for 2, 4 or 6 weeks. The biomass of fish in each aquarium was measured before the experiment and the daily ration was adjusted accordingly after each sampling. Five fish of each -tocopherol feeding group were randomly sampled after 2, 4 and 6 weeks of treatment. The specimens were kept 24 h without feeding before sampling. Head-kidney leucocytes were isolated from each fish, as described above, and the natural cytotoxic activity determined as described below. TARGET CELLS
Cells from the L-1210 line (mouse lymphoma, ATCC CCL-219) were used as targets in the cytotoxic assays. These were cultured in RPMI-1640
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culture medium supplemented with 10% FCS, 100 iu ml 1 penicillin, 100 g ml 1 streptomycin and 2 mM glutamine (Gibco) and incubated at 37 C, 85% relative humidity and 5% CO2 atmosphere and maintained in exponential growth. 3,3 dioctadecyloxacarbocyanine perchlorate (DiO, Sigma) was resuspended in chloroform (Sigma) and stored at 20 C. To label with DiO, the cells were seeded in Petri dishes and incubated in RPMI-1640 culture medium with 10 g ml 1 of DiO for 3 h in a light protected microenvironment. After labelling, free DiO was removed by washing three times in PBS and the staining uniformity was examined.
CYTOTOXIC ASSAYS
The natural cytotoxic activity of gilthead seabream head-kidney leucocytes was studied using a flow cytometric technique based on a double-fluorescent labelling (Cuesta et al., 1999). Each cytotoxic assay was carried out in duplicate. The natural cytotoxic activity of gilthead seabream head-kidney leucocytes (e#ectors) after in vitro incubation with vitamin E supplemented culture medium or from fish fed vitamin E supplemented diet was evaluated. For this, the leucocytes were transferred to 5 ml tubes (Falcon, Becton Dickinson), to which 50 l of DiO-labelled L-1210 cells (targets) (106 cells ml 1 in supplemented RPMI-1640) were added (final e#ector:target ratio of 50:1). The samples were centrifuged (400g, 2 min, 22 C) and incubated at 22 C for 2 h. Samples incubated for 0 h were used as controls to determine initial target viability. The viability of targets maintained at 22 C in sRPMI-1640 culture medium for 2 h was also monitored. At the end of the incubation period, 30 l of propidium iodide (400 g ml 1, Sigma) were added to each sample and mixed gently before analysis in a FACScan (Becton Dickinson) flow cytometer adjusted to obtain optimal discrimination of the target cell population. Data were collected in the form of two-parameter side scatter (granularity) (SSC), forward scatter (size) (FSC), green fluorescence (DiO) (FL1) and red fluorescence (propidium iodide) (FL2) dot plots and histograms obtained by a computerised system. Each analysis was performed on 3000 cells, which were acquired at a rate of 300 cells s 1. The fluorescence histograms represented the relative fluorescence on a logarithmic scale. Standard samples of DiO-labelled target cells or headkidney leucocytes were included in each cytotoxic assay. The FACS only accepted the positive FL1 region, which corresponded to DiO-labelled target cells. The percentage of dead or non-viable target cells showing green and red fluorescence was related with the cytotoxic activity of gilthead seabream leucocytes. The percentage of non-viable target cells after the cytotoxic assay, a parameter which describes the cytotoxic activity of leucocytes, was calculated by the formula: Percentage of non-viable target cells=100(%sample %control)/(100%control).
Percentage of non-viable target cells
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80 70 60
* * * *
* * *
0.01
0.1
*
*
*
50 40 30 20 10 0
1
10 –1
Vitamin E ( µ g ml )
Fig. 1. Natural cytotoxic activity of gilthead seabream head-kidney leucocytes after incubation in vitro with the vitamin E supplemented RPMI-1640 culture medium. Incubation time of 6 h ( ), 24 h ( ) and 48 h (). Horizontal line represents the control value (leucocytes incubated without vitamin E). Data represent the mean S.E. (n=6). *Denote statistically significant di#erences (P<0·05) between control and vitamin E incubated leucocytes. STATISTICAL ANALYSIS
A quantitative study of the flow cytometric results was made using the statistical option of the Lysis software Package (Becton Dickinson). Data were represented as means ( S.E.) and analysed by one-way analysis of variance (ANOVA) and the unpaired Student’s t-test. When the ANOVA test gave di#erences statistically significant (P<0·05) between groups (control and vitamin E), Tukey’s comparison of means test was applied. III. Results IN VITRO EFFECT OF VITAMIN E
The viability of seabream head-kidney leucocytes remained una#ected after acetone or vitamin E-supplemented culture medium treatment. The leucocytes showed changes in their natural cytotoxic activity after incubation with di#erent concentrations of vitamin E. All the vitamin E concentrations assayed significantly enhanced the natural cytotoxic activity of leucocytes (Fig. 1), although higher vitamin E concentrations or longer incubation times (48 h) did not result in greater increases. IN VIVO EFFECT OF VITAMIN E
The natural cytotoxic activity of gilthead seabream head-kidney leucocytes was a#ected by feeding specimens with vitamin E supplemented diets (Fig. 2). After 2 weeks of treatment, the natural cytotoxic activity was only enhanced to a statistically significant degree (P<0·05) with the highest vitamin E supplement dosage assayed (1·8 g kg 1 diet), to reach a value which was almost three times higher than that found in control fish. However, after
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90 80
* *
70 60 50 40 30 20 10 0
2
4
6
Time (weeks)
Fig. 2. Natural cytotoxic activity of gilthead seabream head-kidney leucocytes from specimens fed vitamin E supplemented diets. Vitamin E (g kg 1 diet): 0·1 (control) ( ), 0·7 ( ), 1·3 ( ), 1·9 (). Data represent the mean S.E. (n=6). *Denotes statistically significant di#erences (P<0·05) between control and vitamin E fed groups.
4 weeks of treatment, natural cytotoxic activity was significantly enhanced (P<0·05) even with the lowest vitamin E supplement dosage assayed (0·6 g kg 1 diet). No e#ect of the vitamin E supplemented diets on seabream leucocyte natural cytotoxic activity was observed after 6 weeks. IV. Discussion It has been shown that high vitamin E levels administered in vitro or in vivo can stimulate humoral and cellular immune responses. Vitamin E also decreases induced chemical carcinogenesis, lung influenza-virus in aged animals and the pathogenesis of autoimmune disorders (Haber & Wissler, 1962; Meydani et al., 1986; Hayek et al., 1997). However, very few studies have evaluated the impact of this vitamin on cytotoxic activity (Moriguchi et al., 1990; Ndiweni & Finch, 1995). The e#ect of vitamin E on the fish immune system has also been little studied and the data which have been obtained are controversial (Landolt, 1989; Blazer, 1992; Fletcher, 1997). In in vivo studies, vitamin E supplemented diets increased the phagocytic activity of Atlantic salmon, although the humoral response to bacteria, lymphokine production and respiratory burst remained una#ected (Hardie et al., 1990). Vitamin E supplemented diets in gilthead seabream were correlated with increased complement and phagocytic activites, although neither leucocyte migration or respiratory burst were a#ected (Ortun˜ o et al., 2000). D--tocopherol acetate is one of the most commonly used vitamin E supplements in fish feed, because it was demonstrated that fish possess hydrolytic and transport mechanisms for vitamin E similar to those present in terrestrial animals. Thus, after administration of D--tocopherol acetate, fish can use and store it as free -tocopherol. Furthermore, D--tocopherol acetate was used in
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the present study because it is more stable and shows similar biological activity to D--tocopherol (Hung et al., 1982). Natural cytotoxic activity in fish is a property of natural or non-specific cytotoxic cells (NCC) which are functionally analogous, but which possess di#erent morphological features, to mammalian natural killer cells (Evans et al., 1988). Fish NCC may represent a heterogeneous population of cells involving at least two cell types which show the morphological features of monocytes and lymphocytes (Ellis, 1998). In gilthead seabream, the NCC fit the morphological pattern described for lymphocytes, monocyte-macrophages and acidophilic granulocytes (Meseguer et al., 1994, 1996, 1999; Mulero et al., 1994; Cuesta et al., 1999). The natural cytotoxic activity of seabream head-kidney leucocytes incubated with vitamin E-supplemented culture medium was enhanced for all the concentrations and incubation times assayed including 6 h. However, no statistically significant di#erence was observed in the cytotoxic activity when the di#erent assayed incubation times were compared. The non-statistical di#erences in the 24 h assays between control and 1 or 10 g ml 1 of vitamin E-treated leucocytes could be due to fish variability. In previous studies on the same fish species, neither the phagocytic or the respiratory burst activities of head-kidney leucocytes were significantly enhanced as a consequence of in vitro incubation with vitamin E (Mulero et al., 1998). Previously, it has also been shown that mouse peritoneal macrophages treated in vitro with -tocopherol or calf neutrophils treated with -tocopherol acetate, incorporate and accumulate -tocopherol (Baoutina et al., 1998; Eicher et al., 1994). However, there are no studies comparing possible di#erences between the e#ects of -tocopherol and -tocopherol acetate. When gilthead seabream specimens were fed vitamin E supplemented diets, the natural cytotoxic activity of head-kidney leucocytes was enhanced. According to the data previously obtained by Ortun˜ o et al. (2000), who used the same vitamin E supplementation in the diet and the same experimental times, the enhancement of natural cytotoxic activity was greater and faster than the increases observed in phagocytosis, another non-specific cellular activity. Natural cytotoxic activity was significantly enhanced after 2 weeks of treatment with the highest assayed vitamin E concentration. However, phagocytic activity was enhanced after 4 or 6 weeks of treatment when the middle concentration (1200 mg vitamin E supplemented kg 1 diet) was used. The highest increase observed in natural cytotoxic activity and phagocytosis were almost three- and two-fold, respectively. Serum vitamin E levels, as determined by HPLC, were from 10 g ml 1 (in control specimens) to 88 g ml 1 (in specimens fed 1800 mg vitamin E supplement kg 1 diet) (Ortun˜ o et al., 2000). A correlation seems to exist between the serum vitamin E level and natural cytotoxic activity. Maximum stimulation of the natural cytotoxic activity corresponded with a vitamin E level in serum of 30 g ml 1, while higher vitamin E values correlated with a decreased natural cytotoxic activity, a response which may be due to adaptation to the high vitamin E level in serum. Specimens fed the lowest vitamin E concentration reached this serum vitamin E level after 4 weeks, at the same time as the cytotoxic activity was seen to increase. Similar findings regarding the adaptation of fish immune
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system cells to high vitamin E serum levels were observed for T and B lymphocytes (Verlhac et al., 1993). The great di#erence in the immunomodulatory e#ect of vitamin E on natural cytotoxic activity and on other humoral or cellular immune responses in teleosts might be due to vitamin E location (mainly in cell membranes), which could explain the absence of a direct e#ect of the high vitamin E dosage on humoral factors. However, the di#erences observed between di#erent cellular activities could be due to other factors, such as the status of vitamin E in cell membranes, which may a#ect, directly or indirectly, the membranebound receptor-mediated communication between cells, protein and/or lipid mediators and glycoconjugate biosynthesis (Blumberg, 1993). Furthermore, the activation of alveolar macrophages by macrophage-activating factor produced by lymphocytes has been demonstrated in rats fed with high dietary doses of vitamin E (Moriguchi et al., 1990). Perhaps, similar routes of NCC activation involving cytokines are operating in fish although further studies are needed to clarify this. To conclude, the present results indicate that vitamin E increases the in vitro and in vivo natural cytotoxic activity of gilthead seabream headkidney leucocytes against tumor cells. In vitro, all the vitamin E concentrations and incubation times assayed produced similar increases in natural cytotoxic activity. In vivo, this e#ect was observed after 2 or 4 weeks of treatment using a high (1·8 g vitamin E supplemented kg 1 diet) or low (600 mg vitamin E supplemented kg 1 diet) concentration of vitamin, respectively. The immunostimulant e#ect of this vitamin on natural cytotoxic activity was not observed after 6 weeks, which suggests adaptation of this activity to the high level of vitamin. This work was supported by a project P.E.T.R.I. (reference number 95-0051-0P) and another from ‘Fundación Séneca, Centro de Coordinación de la Investigación’ (reference number PB/9/FS/97) of Murcia (Spain). Mr A. Cuesta, Mr J. Ortun˜ o and Dr M. A. Esteban have grants from the Spanish Ministry of Education and Culture and the ‘Fundación Séneca, Centro de Coordinación de la Investigación’, respectively.
References Baoutina, A., Dean, R. T. & Jessup, W. (1998). -tocopherol supplementation of macrophages does not influence their ability to oxidise LDL. Journal of Lipid Research 39, 114–130. Beharka, A., Redican, S., Leka, L. & Meydani, S. N. (1997). Vitamin E status and immune function. In Methods in Enzymology: Vitamins and Coenzymes, Part L (D. B. McCormick, J. W. Suttie & C. Wagner, eds) pp. 247–263. New York: Academic Press. Blazer, V. S. (1992). Nutrition and disease resistance in fish. Annual Review of Fish Diseases 2, 309–323. 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. Blumberg, J. B. (1993). The role of vitamin E in immunity during aging. In Vitamin E, Its Usefulness in Health and in Curing Diseases (M. Mini, H. Nakamura & A. T. Diplock, eds) pp. 219–229. Tokio: S. Karger Publishing.
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Cowey, C. B., Adron, J. W., Walton, M. J., Murray, J., Youngson, A. & Knox, D. (1981). Tissue distribution, uptake, and requirement for -tocopherol of rainbow trout (Salmo gairdneri) fed diets with a minimal content of unsaturated fatty acids. Journal of Nutrition 111, 1556–1567. Cuesta, A., Esteban, M. A. & Meseguer, J. (1999). Natural cytotoxic activity of gilthead seabream (Sparus aurata L.) leucocytes. Assessment by flow cytometry and microscopy. Veterinary Immunology and Immunopathology 71, 161–171. Eicher, S. D., Morrill, J. L. & Blecha, F. (1994). Vitamin concentration and function of leucocytes from dairy calves supplemented with vitamin A, vitamin E, and -carotene in vitro. Journal of Dairy Science 77, 560–565. Ellis, A. E. (1998). Fish immune system. In Encyclopedia of Immunology (P. J. Delves & I. M. Roitt, eds) pp. 920–926. London: Academic Press. 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. Evans, D. L., Jaso-Friedman, L., Smith, E. E. Jr, John, A. St., Koren, H. S. & Harris, D. T. (1988). Identification of a putative antigen receptor on fish nonspecific cytotoxic cells with monoclonal antibodies. The Journal of Immunology 141, 324–332. Fletcher, T. C. (1997). Dietary e#ects on stress and health. In Fish stress and health in aquaculture (G. K. Iwama, A. D. Pickering, J. P. Sumpter & C. B. Schreck, eds) pp. 223–246. Cambridge: Cambridge University Press. Haber, S. L. & Wissler, R. W. (1962). E#ect of vitamin E on carcinogenicity of methylcholanthrene. Proceedings of the Society for Experimental Biology & Medicine 111, 774–775. 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. Hayek, M. G., Taylor, S. F., Bender, B. S., Han, S. N., Meydani, M., Smith, D. E., Eghtesada, S. & Meydani, S. N. (1997). Vitamin E supplementation decreases lung virus titers in mice infected with influenza. Journal of Infection Disease 176, 273–276. Hung, S. O., Moon, T. W., Hilton, J. W. & Slinger, S. J. (1982). Uptake, transport and distribution of dl--tocopheryl acetate compared to d--tocopherol in rainbow trout (Salmo gairdneri). Journal of Nutrition 112, 1590–1599. Landolt, M. L. (1989). The relationship between diet and the immune response of fish. Aquaculture 79, 193–206. Meseguer, J., Esteban, M. A., Lopez-Ruiz, A. & Bielek, E. (1994). Ultrastructure of nonspecific cytotoxic cells in teleosts. I. E#ector-target cell binding in a marine and a freshwater species (Seabream: Sparus aurata L., and Carp: Cyprinus carpio L.). Anatomical Record 239, 468–474. Meseguer, J., Esteban, M. A. & Mulero, V. (1996). Nonspecific cell-mediated cytotoxicity in the seawater teleosts Sparus aurata and Dicentrarchus labrax: Ultrastructure study of target cell death mechanisms. Anatomical Record 244, 499–505. Meseguer, J., Esteban, M. A. & Mulero, V. (1999). Natural cytotoxic response in fish: A review. In Ichthyology. Recent Research Advances (D. N. Saksena, ed.) pp. 195–208. Enfield: Science Publishers Inc. Meydani, S. N., Meydani, M., Verdon, C. P., Shapiro, A. A., Blumberg, J. B. & Hayes, K. C. (1986). Vitamin E supplementation suppresses prostaglandin E2 synthesis and enhances the immune response of aged mice. Mechanisms of Ageing Development 34, 191–201. Moriguchi, S., Kobayshi, N. & Kishino, Y. (1990). High dietary intakes of vitamin E and cellular immune function in rats. Journal of Nutrition 120, 1096–1102. Mulero, V., Esteban, M. A., Mun˜ oz, J. & Meseguer, J. (1994). Non-specific cytotoxic response against tumor target cells mediated by leucocytes from seawater teleosts, Sparus aurata and Dicentrarchus labrax: An ultrastructural study. Archives of Histology and Cytology 57, 351–358.
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Mulero, V., Esteban, M. A. & Meseguer, J. (1998). E#ects of in vitro addition of exogenous vitamins C and E on gilthead seabream (Sparus aurata L.) phagocytes. Veterinary Immunology and Immunopathology 66, 185–199. Ndiweni, N. & Finch, J. M. (1995). E#ects of in vitro supplementation of bovine mammary gland macrophages and peripheral blood lymphocytes with -tocopherol and sodium selenite: Implications for udder defences. Veterinary Immunology and Immunopathology 47, 111–121. 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. Tu, K. Y., Matthews, R. & Matthews, K. S. (1995). Protective e#ect of vitamin E on lymphocyte growth capacity during incubation in vitro. Mechanisms of Ageing Development 82, 129–148. 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.
Vitamin E increases natural cytotoxic activity in seabream (Sparus aurata L.) A. CUESTA, M. A. ESTEBAN, J. ORTUÑO AND J. MESEGUER* Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain (Received 27 March 2000, accepted after revision 9 October 2000) The natural cytotoxic activity of head-kidney leucocytes from gilthead seabream (Sparus aurata L.), after in vitro and in vivo vitamin E treatment, against tumor cells was studied by flow cytometry. Leucocytes were incubated in culture medium with di#erent vitamin E supplementations (0·01–10 g ml 1) for 6, 24 or 48 h and the results demonstrate that all the assayed vitamin E supplementations significantly enhanced the natural cytotoxic activity of leucocytes. To determine the e#ect of a high dietary level of vitamin E on this activity, fish were fed with 0 (control), 600, 1200 or 1800 mg of vitamin E supplementation kg 1 diet for 2, 4 or 6 weeks. After 2 and 4 weeks of treatment, the natural cytotoxic activity was significantly enhanced at the highest (1·8 g kg 1 diet) and lowest (600 mg kg 1 diet) vitamin E supplement dosage, respectively. No e#ect of the vitamin E supplemented diet on seabream leucocyte natural cytotoxic cell activity was observed after 6 weeks of treatment. 2001 Academic Press Key words:
vitamin E, natural cytotoxic activity, non-specific cytotoxic cells (NCC), leucocytes, gilthead seabream (Sparus aurata L.).
I. Introduction Vitamin E, besides being a very important lipid-soluble micronutrient (Tu et al., 1995), is a normal constituent of all cellular membranes, being particularly abundant in immune cell membranes, where it is an essential component for cell functioning. Furthermore, due to its antioxidant properties, vitamin E interacts with other nutrients, especially polyunsaturated fatty acids (Beharka et al., 1997). The role played by vitamin E in several immunological responses has been studied in mammals (Meydani et al., 1986; Moriguchi et al., 1990; Ndiweni & Finch, 1995; Beharka et al., 1997) and teleosts (Cowey et al., 1981; Hung et al., 1982; Blazer & Wolke, 1984; Hardie et al., 1990; Mulero et al., 1998; Ortun˜o et al., 2000). Mammals fed vitamin E-depleted diets showed depressed immune activities such as resistance to infection, specific antibody responses, splenic antibody forming cells, in vitro mitogenic responses of lymphocytes, reticuloendothelial system clearance or phagocytic index. All these immune functions were enhanced as a consequence of -tocopherol intake (Meydani & *Corresponding author. E-mail: [email protected] 1050–4648/01/040293+10 $35.00/0
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Blumberg, 1992). The available data on the e#ect of vitamin E on the fish immune system are particularly scarce although some similarities with mammals may be observed. For example, humoral immune responses, such as complement activity and haemagglutination and haemolysin titres, were depressed in trout fed vitamin-E depleted diets (Blazer & Wolke, 1984), while Atlantic salmon fed a vitamin E-supplemented diet showed una#ected humoral responses (Hardie et al., 1990). Similarly, T and B cell responses and phagocytic activity were depressed in trout fed vitamin E-depleted diets (Blazer & Wolke, 1984), while Atlantic salmon fed vitamin E-supplemented diet exhibited increased phagocytic activity although neither their respiratory burst activity or their lymphokine production were a#ected (Hardie et al., 1990). Moreover, in our laboratory we have previously described both the in vitro and in vivo e#ect of vitamin E (-tocopherol) on the gilthead seabream non-specific immune response, particularly with respect to leucocyte migration and phagocytic and respiratory burst activities (Mulero et al., 1998; Ortun˜ o et al., 2000). Although natural cytotoxic cell activity is a major immune defence mechanism against virus-infected cells, tumor cells and parasites, no data exist about the e#ect of vitamin E on the fish cytotoxic cell response. The aim of the present work was to study the in vitro and in vivo e#ect of vitamin E on gilthead seabream leucocyte natural cytotoxic activity. II. Materials and Methods ANIMALS
One hundred 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 a running seawater aquaria (flow rate 1500 l h 1) at 20 C with a natural photoperiod. Animals were fed a commercial pellet diet (ProAqua S.A., Palencia, Spain) at a rate of 1·5% body weight day 1.
HEAD-KIDNEY LEUCOCYTE ISOLATION
The animals were anaesthetized with 100 mg l 1 MS222 (Sandoz) and the head-kidney leucocytes were isolated under sterile conditions. Briefly, the 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 nylon mesh (mesh size 102 m). Head-kidney cell suspensions were layered over a 48% Percoll density gradient (Pharmacia) and centrifuged at 400g for 30 min at 4 C (Esteban et al., 1998). After centrifugation, the band of leucocytes above the 48% interface were collected with a Pasteur pipette and washed twice. Cell
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viability was higher than 98%, as determined by the trypan blue exclusion test. VITAMIN E SUPPLEMENTED CULTURE MEDIUM
Vitamin E was prepared by dissolving -tocopherol acetate (Sigma) in acetone (10 mg ml 1). This solution was diluted in sRPMI culture medium to obtain a 0·1 mg ml 1 vitamin E stock solution, which was aliquoted and stored at 80 C until use. To study the in vitro e#ect of vitamin E on the head-kidney leucocyte natural cytotoxic activity, previously isolated leucocytes were adjusted to 5·55106 cells ml 1 in sRPMI-1640 medium supplemented with 10% fetal calf serum (FCS). Then, 450 l of the leucocyte suspensions were dispensed into the wells of a flat bottomed 48 well plate (Nunc). Fifty l of the vitamin E stock solution or 10 times serially diluted stock solution were added to each well, to give a final vitamin E concentration ranging from 0·01–10 g ml 1. Fifty l of culture medium were used for the controls. The samples were incubated at 22 C for 6, 24 or 48 h. The viability of leucocytes incubated with acetone or di#erent vitamin E-supplemented culture medium was also monitored by flow cytometry. VITAMIN E SUPPLEMENTED DIETS
Four experimental diets were prepared in the laboratory from a commercial pellet diet (-tocopherol content 100 mg kg 1). For this, three vitamin E solutions of 24, 48 and 72 mg -tocopherol acetate (Sigma) ml 1 fish oil were made. 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 600, 1200 and 1800 mg vitamin E kg 1 diet. The nonsupplemented diet (control) was prepared by spraying the feed with fish oil alone. The -tocopherol level of the fish oil used to dilute the vitamin E was less than 1 g ml 1, as determined by high-pressure-liquid-chromatography (HPLC) (Ortun˜ o et al., 2000). FISH: TREATMENT AND SAMPLING
The fish were divided randomly into four groups, which were distributed in 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%) each day for 2, 4 or 6 weeks. The biomass of fish in each aquarium was measured before the experiment and the daily ration was adjusted accordingly after each sampling. Five fish of each -tocopherol feeding group were randomly sampled after 2, 4 and 6 weeks of treatment. The specimens were kept 24 h without feeding before sampling. Head-kidney leucocytes were isolated from each fish, as described above, and the natural cytotoxic activity determined as described below. TARGET CELLS
Cells from the L-1210 line (mouse lymphoma, ATCC CCL-219) were used as targets in the cytotoxic assays. These were cultured in RPMI-1640
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culture medium supplemented with 10% FCS, 100 iu ml 1 penicillin, 100 g ml 1 streptomycin and 2 mM glutamine (Gibco) and incubated at 37 C, 85% relative humidity and 5% CO2 atmosphere and maintained in exponential growth. 3,3 dioctadecyloxacarbocyanine perchlorate (DiO, Sigma) was resuspended in chloroform (Sigma) and stored at 20 C. To label with DiO, the cells were seeded in Petri dishes and incubated in RPMI-1640 culture medium with 10 g ml 1 of DiO for 3 h in a light protected microenvironment. After labelling, free DiO was removed by washing three times in PBS and the staining uniformity was examined.
CYTOTOXIC ASSAYS
The natural cytotoxic activity of gilthead seabream head-kidney leucocytes was studied using a flow cytometric technique based on a double-fluorescent labelling (Cuesta et al., 1999). Each cytotoxic assay was carried out in duplicate. The natural cytotoxic activity of gilthead seabream head-kidney leucocytes (e#ectors) after in vitro incubation with vitamin E supplemented culture medium or from fish fed vitamin E supplemented diet was evaluated. For this, the leucocytes were transferred to 5 ml tubes (Falcon, Becton Dickinson), to which 50 l of DiO-labelled L-1210 cells (targets) (106 cells ml 1 in supplemented RPMI-1640) were added (final e#ector:target ratio of 50:1). The samples were centrifuged (400g, 2 min, 22 C) and incubated at 22 C for 2 h. Samples incubated for 0 h were used as controls to determine initial target viability. The viability of targets maintained at 22 C in sRPMI-1640 culture medium for 2 h was also monitored. At the end of the incubation period, 30 l of propidium iodide (400 g ml 1, Sigma) were added to each sample and mixed gently before analysis in a FACScan (Becton Dickinson) flow cytometer adjusted to obtain optimal discrimination of the target cell population. Data were collected in the form of two-parameter side scatter (granularity) (SSC), forward scatter (size) (FSC), green fluorescence (DiO) (FL1) and red fluorescence (propidium iodide) (FL2) dot plots and histograms obtained by a computerised system. Each analysis was performed on 3000 cells, which were acquired at a rate of 300 cells s 1. The fluorescence histograms represented the relative fluorescence on a logarithmic scale. Standard samples of DiO-labelled target cells or headkidney leucocytes were included in each cytotoxic assay. The FACS only accepted the positive FL1 region, which corresponded to DiO-labelled target cells. The percentage of dead or non-viable target cells showing green and red fluorescence was related with the cytotoxic activity of gilthead seabream leucocytes. The percentage of non-viable target cells after the cytotoxic assay, a parameter which describes the cytotoxic activity of leucocytes, was calculated by the formula: Percentage of non-viable target cells=100(%sample %control)/(100%control).
Percentage of non-viable target cells
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80 70 60
* * * *
* * *
0.01
0.1
*
*
*
50 40 30 20 10 0
1
10 –1
Vitamin E ( µ g ml )
Fig. 1. Natural cytotoxic activity of gilthead seabream head-kidney leucocytes after incubation in vitro with the vitamin E supplemented RPMI-1640 culture medium. Incubation time of 6 h ( ), 24 h ( ) and 48 h (). Horizontal line represents the control value (leucocytes incubated without vitamin E). Data represent the mean S.E. (n=6). *Denote statistically significant di#erences (P<0·05) between control and vitamin E incubated leucocytes. STATISTICAL ANALYSIS
A quantitative study of the flow cytometric results was made using the statistical option of the Lysis software Package (Becton Dickinson). Data were represented as means ( S.E.) and analysed by one-way analysis of variance (ANOVA) and the unpaired Student’s t-test. When the ANOVA test gave di#erences statistically significant (P<0·05) between groups (control and vitamin E), Tukey’s comparison of means test was applied. III. Results IN VITRO EFFECT OF VITAMIN E
The viability of seabream head-kidney leucocytes remained una#ected after acetone or vitamin E-supplemented culture medium treatment. The leucocytes showed changes in their natural cytotoxic activity after incubation with di#erent concentrations of vitamin E. All the vitamin E concentrations assayed significantly enhanced the natural cytotoxic activity of leucocytes (Fig. 1), although higher vitamin E concentrations or longer incubation times (48 h) did not result in greater increases. IN VIVO EFFECT OF VITAMIN E
The natural cytotoxic activity of gilthead seabream head-kidney leucocytes was a#ected by feeding specimens with vitamin E supplemented diets (Fig. 2). After 2 weeks of treatment, the natural cytotoxic activity was only enhanced to a statistically significant degree (P<0·05) with the highest vitamin E supplement dosage assayed (1·8 g kg 1 diet), to reach a value which was almost three times higher than that found in control fish. However, after
298 Percentage of non-viable target cells
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90 80
* *
70 60 50 40 30 20 10 0
2
4
6
Time (weeks)
Fig. 2. Natural cytotoxic activity of gilthead seabream head-kidney leucocytes from specimens fed vitamin E supplemented diets. Vitamin E (g kg 1 diet): 0·1 (control) ( ), 0·7 ( ), 1·3 ( ), 1·9 (). Data represent the mean S.E. (n=6). *Denotes statistically significant di#erences (P<0·05) between control and vitamin E fed groups.
4 weeks of treatment, natural cytotoxic activity was significantly enhanced (P<0·05) even with the lowest vitamin E supplement dosage assayed (0·6 g kg 1 diet). No e#ect of the vitamin E supplemented diets on seabream leucocyte natural cytotoxic activity was observed after 6 weeks. IV. Discussion It has been shown that high vitamin E levels administered in vitro or in vivo can stimulate humoral and cellular immune responses. Vitamin E also decreases induced chemical carcinogenesis, lung influenza-virus in aged animals and the pathogenesis of autoimmune disorders (Haber & Wissler, 1962; Meydani et al., 1986; Hayek et al., 1997). However, very few studies have evaluated the impact of this vitamin on cytotoxic activity (Moriguchi et al., 1990; Ndiweni & Finch, 1995). The e#ect of vitamin E on the fish immune system has also been little studied and the data which have been obtained are controversial (Landolt, 1989; Blazer, 1992; Fletcher, 1997). In in vivo studies, vitamin E supplemented diets increased the phagocytic activity of Atlantic salmon, although the humoral response to bacteria, lymphokine production and respiratory burst remained una#ected (Hardie et al., 1990). Vitamin E supplemented diets in gilthead seabream were correlated with increased complement and phagocytic activites, although neither leucocyte migration or respiratory burst were a#ected (Ortun˜ o et al., 2000). D--tocopherol acetate is one of the most commonly used vitamin E supplements in fish feed, because it was demonstrated that fish possess hydrolytic and transport mechanisms for vitamin E similar to those present in terrestrial animals. Thus, after administration of D--tocopherol acetate, fish can use and store it as free -tocopherol. Furthermore, D--tocopherol acetate was used in
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the present study because it is more stable and shows similar biological activity to D--tocopherol (Hung et al., 1982). Natural cytotoxic activity in fish is a property of natural or non-specific cytotoxic cells (NCC) which are functionally analogous, but which possess di#erent morphological features, to mammalian natural killer cells (Evans et al., 1988). Fish NCC may represent a heterogeneous population of cells involving at least two cell types which show the morphological features of monocytes and lymphocytes (Ellis, 1998). In gilthead seabream, the NCC fit the morphological pattern described for lymphocytes, monocyte-macrophages and acidophilic granulocytes (Meseguer et al., 1994, 1996, 1999; Mulero et al., 1994; Cuesta et al., 1999). The natural cytotoxic activity of seabream head-kidney leucocytes incubated with vitamin E-supplemented culture medium was enhanced for all the concentrations and incubation times assayed including 6 h. However, no statistically significant di#erence was observed in the cytotoxic activity when the di#erent assayed incubation times were compared. The non-statistical di#erences in the 24 h assays between control and 1 or 10 g ml 1 of vitamin E-treated leucocytes could be due to fish variability. In previous studies on the same fish species, neither the phagocytic or the respiratory burst activities of head-kidney leucocytes were significantly enhanced as a consequence of in vitro incubation with vitamin E (Mulero et al., 1998). Previously, it has also been shown that mouse peritoneal macrophages treated in vitro with -tocopherol or calf neutrophils treated with -tocopherol acetate, incorporate and accumulate -tocopherol (Baoutina et al., 1998; Eicher et al., 1994). However, there are no studies comparing possible di#erences between the e#ects of -tocopherol and -tocopherol acetate. When gilthead seabream specimens were fed vitamin E supplemented diets, the natural cytotoxic activity of head-kidney leucocytes was enhanced. According to the data previously obtained by Ortun˜ o et al. (2000), who used the same vitamin E supplementation in the diet and the same experimental times, the enhancement of natural cytotoxic activity was greater and faster than the increases observed in phagocytosis, another non-specific cellular activity. Natural cytotoxic activity was significantly enhanced after 2 weeks of treatment with the highest assayed vitamin E concentration. However, phagocytic activity was enhanced after 4 or 6 weeks of treatment when the middle concentration (1200 mg vitamin E supplemented kg 1 diet) was used. The highest increase observed in natural cytotoxic activity and phagocytosis were almost three- and two-fold, respectively. Serum vitamin E levels, as determined by HPLC, were from 10 g ml 1 (in control specimens) to 88 g ml 1 (in specimens fed 1800 mg vitamin E supplement kg 1 diet) (Ortun˜ o et al., 2000). A correlation seems to exist between the serum vitamin E level and natural cytotoxic activity. Maximum stimulation of the natural cytotoxic activity corresponded with a vitamin E level in serum of 30 g ml 1, while higher vitamin E values correlated with a decreased natural cytotoxic activity, a response which may be due to adaptation to the high vitamin E level in serum. Specimens fed the lowest vitamin E concentration reached this serum vitamin E level after 4 weeks, at the same time as the cytotoxic activity was seen to increase. Similar findings regarding the adaptation of fish immune
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system cells to high vitamin E serum levels were observed for T and B lymphocytes (Verlhac et al., 1993). The great di#erence in the immunomodulatory e#ect of vitamin E on natural cytotoxic activity and on other humoral or cellular immune responses in teleosts might be due to vitamin E location (mainly in cell membranes), which could explain the absence of a direct e#ect of the high vitamin E dosage on humoral factors. However, the di#erences observed between di#erent cellular activities could be due to other factors, such as the status of vitamin E in cell membranes, which may a#ect, directly or indirectly, the membranebound receptor-mediated communication between cells, protein and/or lipid mediators and glycoconjugate biosynthesis (Blumberg, 1993). Furthermore, the activation of alveolar macrophages by macrophage-activating factor produced by lymphocytes has been demonstrated in rats fed with high dietary doses of vitamin E (Moriguchi et al., 1990). Perhaps, similar routes of NCC activation involving cytokines are operating in fish although further studies are needed to clarify this. To conclude, the present results indicate that vitamin E increases the in vitro and in vivo natural cytotoxic activity of gilthead seabream headkidney leucocytes against tumor cells. In vitro, all the vitamin E concentrations and incubation times assayed produced similar increases in natural cytotoxic activity. In vivo, this e#ect was observed after 2 or 4 weeks of treatment using a high (1·8 g vitamin E supplemented kg 1 diet) or low (600 mg vitamin E supplemented kg 1 diet) concentration of vitamin, respectively. The immunostimulant e#ect of this vitamin on natural cytotoxic activity was not observed after 6 weeks, which suggests adaptation of this activity to the high level of vitamin. This work was supported by a project P.E.T.R.I. (reference number 95-0051-0P) and another from ‘Fundación Séneca, Centro de Coordinación de la Investigación’ (reference number PB/9/FS/97) of Murcia (Spain). Mr A. Cuesta, Mr J. Ortun˜ o and Dr M. A. Esteban have grants from the Spanish Ministry of Education and Culture and the ‘Fundación Séneca, Centro de Coordinación de la Investigación’, respectively.
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