Fish & Shellfish Immunology (1998) 8, 631–638 Article No. fi980172
Alpha-melanocyte stimulating hormone (á-MSH) and melanin-concentrating hormone (MCH) stimulate phagocytosis by head kidney leucocytes of rainbow trout (Oncorhynchus mykiss) in vitro JAMES HARRIS
AND
DAVID J. BIRD*
Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol, BS16 1QY, U.K. (Received 14 July 1998, accepted in revised form 14 September 1998) Alpha-melanocyte stimulating hormone (á-MSH) and melanin-concentrating hormone (MCH) are two peptides with antagonistic roles in the regulation of skin pigmentation in teleost fish. Both have also been implicated in the modulation of the stress response via the hypothalamo-pituitary-adrenal (HPA) axis in fish and other vertebrates. Alpha-MSH is also known to be a potent immunomodulatory peptide in mammals, while both hormones have been shown to influence the immune responses of trout in vitro. Head kidney phagocytes (macrophages and neutrophils) were exposed to á-MSH at concentrations of 0·05 to 10 nM in vitro for 60 min and the phagocytic activity of glass-adherent cells was assessed microscopically. At 1, 5 and 10 nM, á-MSH significantly increased the percentage of phagocytes that ingested heat-killed yeast cells. Percentage phagocytosis was also significantly increased when cells were exposed to 0·1, 1 and 10 nM des-acetyl-á-MSH. When á-MSH and MCH were added to cells together, at concentrations of 1 nM and 50 nM respectively, the stimulatory e#ects of both hormones were diminished. The results suggest that these peptides may play an immunomodulatory role in the 1998 Academic Press fish immune system. Key words:
alpha-melanocyte stimulating hormone, immuno-endocrinology, melanin-concentrating hormone, phagocytosis, rainbow trout.
I. Introduction Alpha-melanocyte stimulating hormone (á-MSH) is a tridecapeptide derived from the precursor molecule, pro-opiomelanocortin (POMC). The peptide is common to all vertebrates and has been highly conserved throughout its evolutionary history. In teleost fish and other lower vertebrates, á-MSH is involved in the control of skin pigmentation. Its secretion from the pituitary gland into the circulation results in the dispersion of melanin granules within dermal melanophores, causing the skin to darken (Eberle, 1988). An antagonistic peptide, melanin-concentrating hormone (MCH), also released from the pituitary gland of teleosts, has the opposite e#ect and results in lightening of the skin (Baker et al., 1986; Baker, 1994). *Corresponding author. Email:
[email protected] 1050–4648/98/080631+08 $30.00/0
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In both fish and in mammals, three forms of á-MSH are present in the neurointermediate lobe of the pituitary gland, which di#er only in the degree of acetylation at the N-terminus (Follénius et al., 1985). In salmonids, des-acetylated á-MSH (des-acetyl-á-MSH) is the most abundant form in the pituitary, while in cyprinids, diacetylated á-MSH predominates (Kawauchi et al., 1984; Follénius et al., 1985). In the blood of both trout and carp however, mono-acetylated á-MSH is the most common (Follénius et al., 1986). Alpha-MSH and MCH have been implicated in a number of other functions in vertebrates. There is evidence that both peptides are involved in the modulation of the hypothalamo-pituitary-adrenal (HPA) axis of fish and mammals (Thody, 1980; Green et al., 1991; Baker, 1994). Moreover, in mammals, á-MSH is a potent neuroimmunomodulator that can inhibit fever and all major forms of inflammation (Lipton & Catania, 1997). The peptide exerts its immunological e#ects both by direct interaction with immunocompetent cells in peripheral tissues and by modulation of neuronal pathways within the central nervous system (Watanabe et al., 1993; Ceriani et al., 1994). In the periphery, á-MSH inhibits the production of some pro-inflammatory cytokines, including interferon ã (IFN-ã) and tumour necrosis factor (TNF) by human monocytes (Lipton & Catania, 1997). The peptide also induces production of the anti-inflammatory cytokine, interleukin 10 (IL-10) by human monocytes (Bhardwaj et al., 1996). We have previously shown that á-MSH can stimulate lymphocyte mitogenesis in rainbow trout, an e#ect that is antagonised by MCH (Harris & Bird, 1997), while MCH stimulates the phagocytic activity of rainbow trout head kidney leucocytes in vitro (Harris et al., 1998). The present study concerns the e#ects of á-MSH and des-acetyl-á-MSH, on the phagocytic activity of rainbow trout head kidney phagocytes. The combined e#ects of á-MSH and MCH have also been investigated. II. Materials and Methods ANIMALS AND REAGENTS
Rainbow trout (Oncorhynchus mykiss), weighing 150–200 g, were obtained from Alderley Trout Ltd., Gloucestershire, U.K. They were kept in 225 l black or white tanks containing filtered, aerated water at a temperature of 122 C and under a light regime of 12 h light/12 h dark. Fish were acclimated to these conditions for at least 2 weeks before use and fed commercial trout pellets until 3 days before an experiment. Stocking densities did not exceed 15 fish per tank. Salmonid melanin-concentrating hormone (MCH) was obtained from Calbiochem-Novabiochem Corp. (Beeston, Nottingham, U.K.). Monoacetylated-á-melanocyte-stimulating hormone (á-MSH), des-acetyl-á-MSH and all other reagents were obtained from Sigma Chemical Company (Poole, Dorset, U.K.). Stock solutions of 0·5 ìM MCH and 0·1 ìM á-MSH and des-acetyl-á-MSH were prepared in 1% acetic acid containing 1% bovine serum albumin (BSA) and stored as aliquots at 80 C. Subsequent dilutions were made using Leibovitz medium (L-15) containing 0·1% heat-inactivated
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foetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) solution. A solution of 1% BSA in 1% acetic acid was used for control experiments.
PREPARATION OF YEAST CELL SUSPENSION
A suspension of yeast cells was prepared by mixing 0·5 g commercial bakers’ yeast (Saccharomyces cerevisiae) in 30 ml 0·01 M phosphate-bu#ered saline (PBS, pH7·4) and boiling for 10 min. The cells were washed three times in PBS and then resuspended in L-15 containing 4% P/S and 0·1% heat-inactivated FBS at a final concentration of 1·2108 cells ml 1.
PHAGOCYTOSIS ASSAY
Head kidney phagocytes were isolated and cultured using a method based on that of Narnaware et al. (1994). Following anaesthetisation with benzocaine (50 mg l 1), fish were bled from the severed caudal blood vessels to reduce erythrocyte contamination. The head kidney was removed and disrupted through a 70 ìm cell strainer into 5 ml L-15 containing 0·1% FBS and 1% P/S. After washing once (1000 g, 10 min), 900 ìl aliquots of the resultant cell suspension (containing 1–5107 viable leucocytes ml 1) were mixed with 100 ìl of hormone solution (or medium alone for the controls). One millilitre of the suspensions was incubated on methanol-cleaned glass slides for 60 min in a humid chamber at 22 C. Non-adherent cells were washed o# with PBS and 1 ml of yeast cell suspension added to the remaining adherent cells and phagocytosis allowed to proceed. After 60 min, the slides were washed with PBS to remove excess yeast, fixed in methanol and stained with May-Grunwald/Giemsa. Slides were air dried and examined under oil immersion. The percentage of cells undergoing phagocytosis (percentage phagocytosis) and the average number of yeast cells engulfed per active phagocyte (phagocytic index) were determined by randomly inspecting 200 cells per slide. Two types of phagocytic cells were identified; macrophages (rounded nuclei and darkly staining cytoplasm) and neutrophils (irregularly shaped nuclei and chromophobic cytoplasm). Macrophages were the most common cell type, contributing 90–95% of the population on the slides. The presence of yeast cells made it di$cult to distinguish between the two cell types, so all cells were counted. Cells from each fish were exposed to each treatment and statistical significance was determined using Student’s t-test for paired data. III. Results EFFECT OF BSA ON PHAGOCYTOSIS
To ensure that the BSA in the hormone solutions did not influence phagocytosis, the protein was added to head kidney phagocytes from 5 to 8 fish at concentrations of 2–10,000 ng ml 1 (concentrations spanning the range of
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Table 1. The e#ects of á-MSH on the phagocytic activity of rainbow trout head kidney leucocytes in vitro. Values are means1 S.E. from 6 (Experiment 1) or 8 (Experiment 2) fish. Cells from each fish were subdivided into four 1 ml aliquots; 1 aliquot per treatment. Asterisks indicate values are significantly higher than corresponding controls (a) Experiment 1 á-MSH (nM)
Percentage phagocytosis
Phagocytic index
0 0·005 0·5 50
81·081·98 85·172·25 90·581·85* 87·921·77*
3·120·36 2·700·33 3·600·40 3·070·15
á-MSH (nM)
Percentage phagocytosis
Phagocytic index
0 0·1 1 10
82·811·98 85·062·26 92·060·85** 91·250·98*
2·850·15 2·790·10 3·090·07 2·830·09
(b) Experiment 2
(*P
