Effects of in vitro addition of exogenous vitamins C and E on gilthead seabream (Sparus aurata L.) phagocytes

https://doi.org/10.1016/S0165-2427(98)00185-8Get rights and content

Abstract

The phagocytic cell function is an important factor of resistance to infection in fish. Although it is known that several nutritional factors affect phagocyte function and that vitamins C and E modulate the vertebrate immune system, there are no data on the direct effect of these vitamins or on their possible synergistic effects on the fish phagocyte functions. We report the in vitro effect of vitamin C and/or E on the functions and ultrastructure of gilthead seabream (Sparus aurata L.) head–kidney phagocytic leukocytes. Head-kidney leukocytes were incubated with different concentrations of vitamins C and/or E for 48 h and then their migration, respiratory burst, phagocytosis and ultrastructure were studied. Leukocyte migration and phagocytosis increased when vitamin C or E were added, while the respiratory burst was synergistically enhanced when both vitamins were used together. Noticeable ultrastructural changes not previously reported were induced by these vitamins in gilthead seabream phagocytes (acidophilic granulocytes and macrophages). These results indicated that vitamins C and E added in vitro modulate the main functions of gilthead seabream phagocytes and induced ultrastructural changes characteristic of cell activation on such cells.

Introduction

Fish, like other vertebrates, respond to infectious agents in both nonspecific and specific ways, although they depend to a much greater extent on the nonspecific response (Anderson, 1992). Fish phagocytes are a core component in the nonspecific defence system and also play a significant role as accessory cells in both the initiation and regulation of immunity (Clem et al., 1985). Phagocyte functions can be affected by several specific nutrients, such as proteins, lipids, vitamins and minerals (Chandra, 1988; Landolt, 1989). These activators of the phagocyte population may act as immunostimulants by simultaneously improving the nonspecific defence mechanisms, and increasing and extending the duration of the specific immune response. Among such nutrients vitamins C and E play a crucial role (Blazer, 1992)

Vitamin C has been shown to be important in complement activity (Sakamoto et al., 1981), antibody responses, and a variety of other immune functions in higher vertebrates (Panush and Delafuente, 1985). It is also believed to be important for neutrophils and lymphocytes, protecting them from oxidative damage (Anderson et al., 1990). In fish, vitamin C was early recognized as a required nutrient (Kitamura et al., 1965) and more recently, some authors have shown that the dietary intake of vitamin C affects complement activity (Li and Lowell, 1985; Hardie et al., 1991), phagocytosis (Li and Lowell, 1985) and antibody levels (Li and Lowell, 1985; Navarre and Halver, 1989).

Vitamin E is essential for both homeotherms and fish. It is the principal lipid soluble antioxidant of cell membranes and protects unsaturated fatty acids from oxidation (Le Grusse and Watier, 1993). Fish tend to contain a high percentage of long chained highly unsaturated fatty acids in cell membranes to maintain membrane fluidity at low body temperatures. Hence, the dietary intake of vitamin E is of primary importance in fish. Numerous studies indicate that vitamin E plays an important role in the immune system of homeotherms, enhancing phagocytosis, lymphocyte proliferation and antibody production (Panush and Delafuente, 1985). Moreover, it acts synergistically with vitamin C to maintain glutathione peroxidase and superoxide dismutase activities (Le Grusse and Watier, 1993). Similarly, dietary levels of vitamin E are also important in fish immunocompetence. Thus, complement activity and phagocytosis decreased in fish fed with diets deficient in vitamin E (Blazer and Wolke, 1984; Hardie et al., 1990).

Although there are a few data suggesting that the use of dietary vitamin C and E in fish improves their natural resistance to infections, the possible mechanisms responsible for this resistance have not been well established and the results reported are often controversial. In addition, the interactions between these two vitamins have not been studied till now. The aim of this paper was, therefore, to study the effect of the in vitro addition of vitamin C and/or vitamin E on the migration, phagocytosis, respiratory burst and ultrastructure of gilthead seabream (Sparus aurata L.) phagocytes in an attempt to determine which of these parameters are modified by these vitamins, and their relative participation in any conferred resistance to infections. These results will facilitate further research into the immunomodulatory effects of vitamins in this commercially important fish species.

Section snippets

Animals

Twenty specimens of 18 months (400 g mean weight), of the hermaphroditic protandrous seawater teleost gilthead seabream (Sparus aurata L.) obtained from CULMAREX S.A. (Murcia, Spain), were kept in 260 l running seawater aquaria (flow rate 1500 l/h) at 20°C with a 12 h light/darkness cycle. Fish were fed with a commercial pellet diet (Purina) at a feeding rate of 1.5 g dry diet/kg biomass of fish/day. This commercial diet contained 500 mg/kg vitamin C and 300 mg/kg vitamin E. The specimens were

Results

The gilthead seabream head–kidney leukocytes above the 34–51% Percoll interface after incubation with different concentrations of vitamin C and/or vitamin E underwent changes in their migratory, phagocytic and respiratory burst activities, as well as in their ultrastructural features. Cell viability was unaffected by the vitamin treatments.

Discussion

Some studies seemed to indicate that the immune system of bony fish is comparable to that of mammals in various aspects, since both possess nonspecific and specific immune responses with cellular and humoral components. However, fish depend more heavily on nonspecific defence mechanisms (Anderson, 1992) and this is especially true for fish living in cool or cold water since the development of a specific immune response is temperature dependent (Avtalion, 1981; Rijkers et al., 1981).

It is known

Acknowledgements

We wish to thank Drs. A. Toranzo and J.L. Barja for providing the bacteria. This work was supported by the C.I.C.Y.T. (reference project AGF94-1360-C03-02 and P.E.T.R.I. reference project 95-0051-OP). Dr. V. Mulero was supported by a grant from Caja Murcia and Dr. M.A. Esteban from Fundación Séneca. Centro de coordinación de la Investigación (Murcia, Spain).

References (36)

  • Y. Li et al.

    Elevated levels of dietary ascorbic acid increase immune responses in channel catfish

    J. Nutr.

    (1985)
  • V. Mulero et al.

    Dietary intake of levamisole enhances the immune response and disease resistance of the marine teleost gilthead seabream (Sparus aurata L.)

    Fish Shellfish Immunol.

    (1998)
  • O. Navarre et al.

    Disease resistance and humoral antibody production in rainbow trout fed high levels of vitamin C

    Aquaculture

    (1989)
  • G.J.E Sharp et al.

    Leucocyte migration in rainbow trout (Onchorhynchus mykiss [Walbaum]): optimization of migration conditions and responses to host and pathogen (Diphyllobothrium dendriticum [Nitzsch]) derived chemoattractants

    Dev. Comp. Immunol.

    (1991)
  • I. Thompson et al.

    The effect of stress on the immune response of Atlantic salmon (Salmo salar L.) fed diets containing different amount of vitamin C

    Aquaculture

    (1993)
  • D.O. Adams et al.

    The cell biology of macrophage activation

    Ann. Rev. Immunol.

    (1984)
  • R. Anderson et al.

    Effects of ascorbate on leukocytes V. Effects of ascorbate and calcium and sodium ascorbate on certain functions of human blood lymphocytes in vitro

    S. Afr. Med. J.

    (1979)
  • R. Anderson et al.

    Vitamin C and cellular immune functions: protection against hypochlorous acid-mediated inactivation of glyceraldehyde-3-phosphate dehydrogenase and ATP generation in human leucocytes as a possible mechanism of ascorbate-mediated immunostimulation

    Ann. New York Acad. Sci.

    (1990)
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