Fish & Shellfish Immunology (1996) 6, 615–617

SHORT COMMUNICATION

Increase in haemolytic activity of serum from rainbowtrout Oncorhynchus mykiss injected with exogenous

growth hormone

MASAHIRO SAKAI1, YOICHIRO KAJITA2, MASANORI KOBAYASHI

2AND

HIROSHI KAWAUCHI2

1Faculty of Agriculture, Miyazaki University, Miyazaki 889-21, Japan and2School of Fisheries Sciences, Kitasato University, Sanriku, Iwate 002-01,

Japan

(Received 8 August 1996, accepted 12 August 1996)

Key words: serum haemolytic activity, growth hormone, rainbow trout.

Among the numerous functions of growth hormone (GH) is its participation inthe control of immune processes in mammals (Nagy et al., 1983; Auernhammer &Strasburger, 1995). It is known that GH directly a#ects immunocompetent cells(macrophages, lymphocytes and natural killer cells). Berczi & Nagy (1987) demon-strated that GH secreted from the adenohypophysis has stimulatory e#ects on bothprimary and secondary lymphoid organs of rat. T-lymphocytes have receptors for GHand prolactin, and several immune responses are regulated by these hormones. GHinjected into fish increases mitogenic responses of lymphocytes (Sakai et al., 1996a) andNK cell activities (Kajita et al., 1992). In addition, Sakai et al. (1995, 1996b) havereported that exogenous GH given to rainbow trout, Oncorhynchus mykiss, increasedthe production of superoxide anion in leucocytes. However, the influence of GH onnon-specific humoral immune parameters is still unclear. The complement system is acomplex of enzymes present in the serum, which has antimicrobial activity due to itslytic properties. The purpose of the present study was to determine whether exogenousGH can a#ect the haemolytic activity of serum from rainbow trout.Rainbow trout each weighing 82·1&9·6 g (mean weight&S.D.) were obtained from

Iwate Fisheries Experimental Station, Japan, and maintained in outdoor tanks withfresh running water at 14) C for 2 weeks and fed a commercial diet twice daily.GH was prepared from chum salmon, O. keta, as described by Kawauchi et al. (1986).

Briefly, native GH (nGH) was isolated by fractionation of alkaline pituitary extracts bycolumn chromatography and salt/isoelectric precipitation. The purity of nGH wasconfirmed by HPLC. Two molecular forms of chum salmon growth hormone, GH I andGH II, were recognized and GH I was used. Recombinant GH I (rGH) which waspurified by column chromatography and salt/isoelectric precipitations as described byKawauchi et al. (1986) was also used. The purity of rGH was also confirmed by HPLC.The rGH was examined for contamination by Escherichia coli lipopolysaccharide by theLimulus amoebocyte lysate assay. No endotoxin was detected. The purified nGH andrGH had also been confirmed to have growth promoting activity in rainbow trout(Kawauchi et al., 1986; Kawauchi & Moriyama, 1988). GH was dissolved in a minimumvolume of 0·001 N NaOH at pH 10. After adjusting the pH to 7·2 with 0·01 N HCl, thesolution was diluted with saline to give a final concentration of 1 mg ml"1. Rainbow

6151050–4648/96/080615+03 $25.00/0 ? 1996 Academic Press Limited

trout were anaesthetised with 0·02% MS-222 and injected intraperitoneally with 10 ìgGH/fish. Control fish were injected with the same volume of saline. Five fish of eachgroup were sampled at 1, 3 and 5 days after injection.Blood samples were collected with syringes from the caudal vein of GH-treated

rainbow trout, and allowed to clot at R.T. for 2 h. The serum complement activity wasdetermined by assaying the alternative complement activity (ACH50) (Yano et al., 1988).The serially diluted sera from five fish injected with GH or saline were incubated with4#107 rabbit red blood cells in 10 mM EGTA (ethylene glycol-bis tetraacetic acid)-10 mM MgCl2-GVB

2+ (pH 7·0) at 20) C for 90 min. The haemolytic activity was measuredat O.D. 414 nm by means of an EIA reader (BioRad) and ACH50 was determined. The dataare expressed as mean&S.D. of the number of experiments performed. They weresubjected to one-way analysis of variance using Kruskal–Wallis ranks, followed by theMann–Whitney two-tail U-test, P<0·05 being taken as the minimum significance level.The e#ect of exogenous GH on the haemolytic activity of serum from rainbow trout

is shown in Table 1. The results are expressed in ACH50 units, which represent thecapacity of serum complement to lyse 50% of rabbit red blood cells. Both exogenousnGH and rGH increased the haemolytic activity of serum, observed 3 and 5 days afterGH-injection compared with the values observed in control fish (P<0·005).In mammals, no stimulation of complement by exogenous GH has been reported. The

present findings, therefore, suggest a new e#ect of GH. The ring dove, Streptopeliarisoria, showed increased haemolytic activities of serum after injection with exogenousprolactin (Barriga et al., 1994). Prolactin and GH are evolved from a single ancestralpeptide (Rand-Weaver & Kawauchi, 1993). Both hormones show immune modulatoryactivity such as increased production of superoxide anion by macrophages, theactivation of natural killer cells and the mitogenic responses of lymphocytes (Kelly,1989; Auernhammer & Strasburger, 1995). Further studies are required at the molecu-lar level to clarify the mechanism of action of GH on the activity of serum complement.

References

Auernhammer, C. J. & Strasburger, C. J. (1995). E#ects of growth hormone and insulin-likegrowth factor I on the immune system. European Journal of Endocrinology 133, 635–645.

Barriga, C., Lopez, R. & Rodriguez, A. B. (1994). Haemolytic and bactericidal activity of serumfrom the ring dove (Streptopelia risoria) after treatment with exogenous prolactin.Journal of Comparative Physiology B 164, 499–502.

Berczi, I. & Nagy, E. (1987). The e#ect of prolactin and growth hormone on hemolymphopoietictissue and immune function. In Hormones and Immunity (I. Berczi & K. Kovacs, eds)pp. 145–171. Lancaster: MTP Press.

Kajita, Y., Sakai, M., Kobayashi, M. & Kawauchi, M. (1992). Enhancement of non-specificcytotoxic activity of lymphocytes in rainbow trout Oncorhynchus mykiss injected withgrowth hormone. Fish & Shellfish Immunology 2, 155–157.

Table 1. Haemolytic activity of serum from rainbow trout injected withgrowth hormone

Serum fromfish treatedwith:

Haemolytic activity (ACH50 units)

Days after injection1 3 5

nGH 350&28 447&36* 515&22*rGH 337&53 411&29* 515&24*Saline 360&43 320&43 351&50

nGH, native growth hormone.rGH, recombinant growth hormone.Each value represents the mean&S.D. of five determinations performed in

duplicate.*P<0·005 with respect to the control value.

616 M. SAKAI ET AL.

Kawauchi, H. & Moriyama, S. (1988). Chum salmon growth hormone: isolation and e#ect ongrowth of juvenile rainbow trout. In New and Innovative Advances in Biology/Engineering with Potential for use in Aquaculture. NOAA Technical Reports 1–6, NMFS70, National Marine Fisheries Service, Seattle.

Kawauchi, H., Moriyama, S., Yashuda, A., Yamaguchi, K., Sirahata, K., Kubota, J. & Hirano, T.(1986). Isolation and chracterization of chum salmon growth hormone. Archives ofBiochemistry and Biophysics 244, 542–552.

Kelly, K. W. (1989). Growth hormone, lymphocytes and macrophages. Biochemical Pharmacology38, 705–713.

Nagy, E., Berczi, I. & Frieser, H. G. (1983). Regulation of immunity in rats by lactogenic andgrowth hormones. Acta Endocrinology 102, 351–357.

Rand-Weaver, M. & Kawauchi, H. (1993). Growth hormone, prolactin and somatolactin: astructural overview. InMolecular Biology Frontiers (P. W. Hochachka & T. P. Mommsen,eds) pp. 39–56. Amsterdam: Elsevier.

Sakai, M., Kobayashi, M. & Kawauchi, H. (1995). Enhancement of chemiluminescent responses ofphagocytic cells from rainbow trout, Oncorhynchus mykiss, injected with growth hor-mone. Fish & Shellfish Immunology 5, 375–379.

Sakai, M., Kobayashi, M. & Kawauchi, H. (1996a). Mitogenic e#ect of growth hormone andprolactin on chum salmon Oncorhynchus keta leucocytes in vitro. Veterinary Immunologyand Immunopathology (In press).

Sakai, M., Kobayashi, M. & Kawauchi, H. (1996b). In vitro activation of fish phagocytic cells bygrowth hormone, prolactin and somatolactin. Journal of Endocrinology (In press).

Yano, T., Hatayama, Y., Matsuyama, H. & Nakao, N. (1988). Titration of the alternativecomplement pathway activity of representative cultured fishes. Nippon Suisan Gakkaishi54, 1049–1054.

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