Effects of Steroids on the Antibody Producing Activity of Lymphocytes in Rainbow Trout

Transcription

1 Fisheries Science 65(6), (1999) Effects of Steroids on the Antibody Producing Activity of Lymphocytes in Rainbow Trout Yayi Hou, Yuzuru Suzuki,* and Katsumi Aida Department of Aquatic Biosciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo , Japan (Received February 8, 1999) The effects of steroid hormones on in vitro antibody producing cells from lymphocytes of the peripheral blood, head kidney, spleen and skin in juvenile rainbow trout were examined by ELISPOT assay. Cortisol, testosterone, estradiol-17 ƒà and 11-ketotestosterone reduced dose-dependently the num ber of cells producing both IgM and specific antibody against TNP-LPS. These steroid hormones also reduced IgM secretion from the head kidney and spleen lymphocytes. These results show that the low immunocompetence with the gonadal maturation is caused by the direct effects of steroid hormones on the antibody producing activity of lymphocytes. Moreover, the results also suggest that steroid hor mones suppress the function of B lymphocytes, since TNP-LPS is thought to be thymus-independent antigen. Key words: rainbow trout, IgM, antibody producing cell, cortisol, testosterone, estradiol-17ƒà and 11-ketotestosterone Salmonids were known to have relatively few antibody producing cells during the spawning season.1,2) Plasma IgM levels decreased during the spawning season, when the sex steroids and cortisol (F) were high in both male and female rainbow trout.3,4) F is known to reduce the secretion of IgM from lympho cytes, the number of antibody producing cells (APC) and disease resistance of salmonid fish.5-9) As Weyts et al.10 reported recently in carp, F may induce apoptosis in salmonid B cells. Since fish leukocytes have glucocorticoid receptors,8,11-13) these regressions might be caused by direct action of F on the B cells via the receptors. Interactions be tween the immune system and the hypothalamo-pituitary interrenal axis are thus well established.14) Testosterone (T) was also found to reduce in vitro plaque-forming response of cells from the anterior kidney of immature chinook salmon.15-17) They showed the exis tence of andorogen receptors of the lymphocytes, 16,11) and found that the receptor shows seasonal changes in imma ture fish. 19) In addition to T, we showed the possibility that other more potent sex steroid horomons for gonadal de velopment in both sex, i.e., estradiol-17ƒà (E2) in female and 11-ketotestosterone (11-KT) in male, have im munosuppresive function, although we have not enough knowledge regarding such functions.4) In vitro monitoring of the appearance of antibody producing cells can make clear whether these steroid hormones have a direct effect on the cells or not. From this background, we made further analyses of the direct effects of F, T, E2, and 11-KT on antibody produc ing cells, after Slater and Schreck15) with some modifica tions, using enzyme-linked immunospot (ELISPOT) assay technique. Cells were incubated with steroid hormones, thereafter the cells producing IgM and the antibody against adminstration of antigen, i.e., IgM producing cells (IPC) and specific antibody producing cells (SAPC), were counted by ELISPOT assay. Experiments on the effect of steroid hormones on the IgM production were also con ducted. Fish Materials and Methods Juvenile rainbow trout used in this experiment were ob tained from a local farm. Mean body weight was 85 }15 g. Sixty fish were reared in 80-liter round plastic tanks, 10 fish per tank, supplied with filtered circulating water. Water temperature was at 14 Ž and photoperiod was at 14L:IOD. Fish were fed once daily with commercial trout pellets at a rate of 1 to 2% body weight. Before the experi ment in April, fish were acclimated for three weeks to the above conditions, to exclude the effect of stress due to transport. TNP-LPS The antigen used for in vitro experiment was trinitrophenol-lipopolysaccharide (TNP-LPS). It was pre pared by the method of Jacobs and Morrison.20) Briefly, 100mg of LPS from Escherichia coli 055:B5 (Sigma) were dissolved in 5ml cacodylate buffer, 0.28 M, ph 6.9, and ad justed to ph 11.5 with 10 N NaOH. Then 60mg 2,4,6- trinitrobenzenesulfonic acid (Sigma) in 5ml buffer was added and the reaction was allowed to proceed for 2 hours at room temperature. The conjugate was dialyzed against saline overnight, and the amount of TNP was determined s pectrophotometri cally. 21) TNP-BSA * Corresponding anther.

2 TNP coupled to bovine serum albumin (TNP-BSA) and used as an antigen for ELISPOT was prepared according to Garvey et al.22) Crystallized and lyophilized BSA (frac tion V, Sigma) was used. Antibody Production and Steroids in Raibow Trout 851 for coating. After washing, the cells for SAPC were placed onto the wells. Antibody bound with TNP at the position of SAPC was stained following ELISPOT procedure for APC reported previously.2) Tissue Culture Media RPMI 1640 supplemented with L-glutamine, 10% heat inactivated fetal calf serum, 100 U/ml penicillin, and 100 ƒê g/ml streptomycin, was used as a tissue culture medium (TCM). Steroid Preparation Stock solutions of steroid hormones used for tissue cul ture eperiments were prepared as follows. F (Wako), T (Wako), E2 (Sigma), and 11-KT (Cosmo Bio), were dis solved in ethanol, then allowed to evaporate completely. The residual steroid hormones were suspended in TCM to a final concentration of 10ƒÊg/ml. The solutions were steri lized with 0.22 ƒêm Millipore filter prior to use. Isolation of Leukocytes Fish were anesthetized deeply with 1000 ppm of 2- phenoxyethanol. Each tissue sample of the head kidney, spleen, peripheral blood and skin was removed from the fish aseptically, and placed in 5 ml TCM on ice. Single cell suspensions of these tissues were made by teasing the tissues, followed by repeated intake and expulsion of the tissues through a 5 ml syringe. Peripheral blood and cell suspensions of the head kidney and spleen were applied to Percoll (Pharmacia) incontinuous density gradient ( ) centrifugation in 800 ~g for 40 min at 4 Ž. Lym phocyte rich fraction at the interface of and was separated. Viable cells were counted by 1 % trypan blue exclusion test. Cells from the skin were used without Percoll density gradient centrifugation. The cell suspen sions of epidermis were adjusted to be 5 ~104 cells/ml for the skin and 5 ~106 cells/ in/ for other tissues. The cell com position of the suspension was checked on smear prepara tion after Rowley,23) and the purity of the lymphocytes was calculated to be 88 to 97% of the cells. Cell Culture for Antibody Producing Cell IPC and SAPC were measured according to Slater and Schreck 15) with some modifications. For measurement of IPC, cells were cultured in 24-well plates under 5%CO2 at 18 Ž for 6 days in the presence or absence of steroid hor mones. For SAPC, 0.8ƒÊg/ml of TNP-LPS as antigen was added to the wells. Concentrations of steroid hormones used for the experiments were decided from the data of the annual changes in steroid levels,3,24-26) 10 ng/ml (low con centration) and 100 ng/ml (high concentration) for F, T and 11-KT and 5 ng/m/ (low concentration) and 50 ng/ml (high concentration) for E2. Nonsteroid-treated wells were regarded as controls. Tests were repeated four times. ELISPOT for IgM Producing Cells (IPC) The method for IgM producing cells was described previ ously.2) ELISPOT for Specific Antibody Producing Cells (SAPC) The wells were added with 100 ƒêl of TNP-BSA solution (carbonate buffer, PH 9.6), and allowed overnight at 4 Ž Amount of IgM Produced by Lymphocytes In Vitro Lymphocytes were separated from head kidney and spleen as described above. The cells were cultured in 24 - well plates under 5%CO2 gas at 18 Ž in the presence of steroid hormones, F, T, 11-KT and E2 at 10, 25, 50, and 100 ng/ml. Nonsteroid-treated wells were used as control. At intervals of 2 days, the culture supernatants were re moved from one third of wells of incubation. IgM concen trations were measured by EIA, as already reported.3) Results were expressed as production of IgM within 2 days by 104 leukocytes of each tissue; production of IgM up to each sampling time within 2 days was calculated from the differences between the amount of IgM at the day and that at 2 days before. Day 0 means initial control. The test was repeated four times. Statistics All data were expressed as mean } SE. Significant differ ences between groups of control and steroid administra tion in number of IPC, SAPC and amount of IgM secre tion were identified by one way analysis of variance (ANOVA) followed by Duncan's multiple range test. Results Effects of Steroid Hormones on IPC Changes in the number of IgM secreting cells are shown in Fig. 1. For the lymphocytes from peripheral blood, F at low concentration significantly lowered the number of IPC to 37% of control, while other steroids at low concentra tion had no significant effects. F, T, E2, and 11-KT at high concentration significantly lowered the number of IPC to respective 4, 22, 39, and 22% of control. For lymphocytes from the head kidney, spleen and skin, no steroids at low concentration significantly reduced the number of IPC, while the steroids at high concentration clearly suppressed the number. T and E2 at high concentration were much more suppressive for the lymphocytes from the head kid ney and skin than from spleen, while 11-KT exhibited stronger inhibition of the head kidney lymphocytes than the cells of spleen and the skin. Effects of Steroid Hormones on SAPC Changes in the number of SAPC are shown in Fig. 2. All steroids at low concentration tended to reduce the num ber of SAPC, although the amount of reduction was not significant. F at high concentration significantly decreased the number of SAPC from all four tissues, while other steroids at high concentration demonstrated immunosup pression in varying degrees on the lymphocytes of the different tissues. T at high concentration significantly reduced the number of SAPC to 4 and 12% of controls in the peripheral blood and spleen, respectively, and com pletely suppressed it in the head kidney and the skin. E2 at high concentration showed significant immunosuppres sion on the head kidney. 11-KT at high concentration lo wered the number of SAPC of peripheral blood and spleen

3 852 Hou et al. Fig. 1. The effects of steroid hormones on the number of IgM producing cells (IPC) in the lymphocytes from the peripheral blood, head kidney, spleen and skin. C, control; F, cortisol; T, testosterone; E2, estradiol-17,6 ; 11-KT, I1-ketotestosterone. Each bar represents the mean } SE. Points marked * and ** are significantly different from control for P<0.05 and P<0.01, respectively. Fig. 2. The effects of steroid hormones on the number of specific antibody producing cells (SAPC) in the lymphocytes from the peripheral blood, head kidney, spleen and skin. C, control; F, cortisol; T, testosterone; E2, estradiol-17ƒà; 11-KT, 11-ketotestosterone. Each bar represents the mean±se. Points marked * and ** are significantly different from control for P<0.05 and P<0.01, respectively. to 3 and 30% respectively, and suppressed it completely in the head kidney and the skin. Effects of Steroid Hormones on the Amount of IgM Production The effects of steroid hormones on the amount of IgM secreted from the head kidney lymphocytes are shown in Fig. 3. F treatment caused significant depression of the IgM secretion on days 4 and 6, but not on day 2. Sex steroid treatments also caused significant suppression in a dose-dependent manner at least on day 6. On day 2 only high levels of T exhibited a significant decrease of IgM secretion. The effects of steroid hormones on the amount of IgM

4 Antibody Production and Steroids in Raibow Trout 853 Fig. 3. The effects of steroid hormones on IgM produced by lymphocytes from the head kidney. Each bar represents the mean }SE. Points marked * and ** are significantly different from control for P<0.05 and P<0.01, respectively. Fig. 4. The effects of steroid hormones on IgM produced by lymphocytes from the spleen. Each bar represents the mean }SE. Points marked * and ** are significantly different from control for P<0.05 and P<0.01, respectively. secreted from the cultured spleen lymphocytes are shown in Fig. 4. F treatment caused significant depression of the IgM secretion on days 4 and 6, while not on day 2 except F at a concentration of 50ng/ml. E2 treatment also caused significant suppression on days 4 and 6. The treatments of T and 11-KT at any doses also caused significant suppres sion on day 6, but not on day 4 except at concentrations of 10 and 100ng/ml of both steroid hormones. On day 2, the treatments of sex steroid hormones exhibited no suppres sion. Discussion The ELISPOT assay used here was a sensitive and reli able method for detecting IPC and SAPC in the rainbow trout. The results in this study demonstrated that F, T, E2 and 11-KT reduced clearly the number of both IPC and SAPC in vitro. These steroid hormones also reduced in vitro IgM secretion from the head kidney and spleen lym phocytes. These results suggest that the low immunocom

5 854 Hou et al. petence with the gonadal maturation is caused by the effects of steroid hormones on the antibody producing ac tivity of lymphocytes. Moreover, the results also suggest that steroid hormones act directly on the B cells to sup press IgM production, since TNP-LPS is thought to be thy mus-independent antigen. However, we can not yet deny the participation of factors derived from other cells such as T cells, since we could not use pure B cell populations for the experiments. The effects of steroid hormones were dose-dependent. This is agreeable with the observations that the IgM and IgM producing cells decreased in reverse of significant in creases in plasma steroid hormones during the spawning season.2,3) Reduced number of antibody producing cells by F in salmonid fish were also reported.7,15,27,28) T also reduced the plaque-forming response in vitro of Chinook salmon.15) In addition to these observations we showed here that the E2 and 11-KT, i.e., the most important sex steroid hormones for respective female and male, are also effective for the reduction of immunocompetence. Fish leukocytes have F and T receptors ,16, 181 The reduced IgM producing activity might be caused by direct action of these steroides on IPC. However, we have not enough knowledge on the leukocytes which have these receptors. It is therefore possible that the function of the hormones are not direct but indrect, via some messenger proteins such as cytokine, since we used a mixture of sever al cell types of leukocytes in the in vitro experiment. The results supplied the evidence that the decrease in mu cus IgM levels is probably related to the reduction of IgM secretion from the local lymphocytes. IgM levels in the skin mucus were not necessarily parallel to those in blood plasma, when steroid hormones were administered in vivo.41 Direct suppression of APC and SAPC in the skin may mean that the origin of mucus IgM is lymphocytes of epidermis,29,30) which are sensitive to plasma steroid hor mones. Howeeeever, it is still necessary to explore whether the plasma IgM diffuses into the skin mucus. T and 11-KT were observed to inhibit the response of lymphocytes to specific antigen (TNP-LPS) in the head kid ney and skin, le not in blood and spleen. T induced sup pression in IgM secretion appeared earlier in the head kid ney lymphocytes than in the splenic lymphocytes. The splenic lymphocytes were more sensitive to E2 than the head kidney lymphocytes. In addition, Trip et a1.271 report ed that the head kidney lymphocytes were only sensitive to F early in induction of the antibody response, where the splenic cells were sensitive to F throughout the culture period. These differences in the sensitivity of the lympho cytes to steroid hormones might be caused by the differ ences in the cell compositions of each tissue in nature. Acknowledgments This work was supported by a Grant-in Aid from the Ministry of Agriculture, Forestry and Fisheries. References 1) A. C. Maule, R. Schrech, C. Slater, M. S. Fizpatrick, and C. B. Shreck: Immune and endocrine responses of adult chinook salmon during freshwater immigration and sexual maturation. Fish Shellfish Immunol., 6, (1996). 2) Y. Y. Hou, Y. Suzuki, and K. Aida: Changes in antibody produc ing cells in response to gonadal maturation in rainbow trout, On corhynchus mykiss. Fisheries Sci., 65, (1999). 3) Y. Suzuki, T. Otaka, S. Sato, Y. Y. Hou, and K. Aida: Reproduc tion related immunoglobulin changes in rainbow trout. Fish Phys iol. Biochem., 17, (1997). 4) Y. Y. Hou, Y. Suzuki, and K. Aida: Effects of steroid hormones on immunoglobulin M (IgM) in rainbow trout, Oncorhynchus mykiss. Fish Physiol. Biochem., 20, (1999). 5) A. D. Pickering and J. Duston: Administration of cortisol to brown trout, Salmo trutta L., and its effect on the susceptibility to Saprolegnia infection and furunculosis. J. Fish Biol., 23, (1983). 6) A. D. Pickering and T. G. Pottinger: Cortisol can increase the sus ceptibility of brown trout, Salmo trutta L., to disease without reduc ing the white blood cell count. J. Fish Biol., 27, (1985). 7) S. L. Kaattari and R. A. Tripp: Cellular mechanism of glucocorti coid immunosuppression in salmon. J. Fish Biol., 31, Suppl. A, (1987). 8) A. G. Maule, C. B. Schreck, and S. L. Kaattari: Changes in the im mune system of coho salmon (Oncorhynchus kisutch) during the parr-to-smolt transformation and after implantation of cortisol, Can. J. Fish. Aquat. Sci., 44, (1987). 9) A. D. Pickering and T. G. Pottinger: Stress responses and disease resistance in salmonid fish: Effects of chronic elevation of plasma cortisol. Fish Physiol. Biochem., 7, (1989). 10) F. A. A. Weytes, G. Flik, J. H. W. M. Rombout, and B. M. L. Ver burg-van Kemenade: Cortisol induces apoptosis in activated B cells, not in other lymphoid cells of the common carp, Cyprinus carpio L. Dev. Comp. Immunol., 22, (1998). 11) A. C. Maule and C. B. Shreck: Glucocorticoid receptors in leuko cytes and gill of juvenile coho salmon (Oncorhynchus kisutch). Gen. Comp. Endocrinol., 77, (1990). 12) A. C. Maule and C. B. Shreck: Stress and cortisol treatment changed affinity and number of glucocorticoid receptors in leuko cytes and gill of coho salmon. Gen. Comp. Endocrinol., 84, (1991). 13) M. Tagawa, H. Hagiwara, A. Takemura, S. Hirose, and T. Hirano: Partial cloning of hormone-binding domain of the cortisol receptor in tilapia, Oreochromis mossambicus, and changes in the mrna level during embryonic development. Gen. Comp. Endocrinol., 108, (1997). 14) F. A. A. Weyts, N. Cohen, G. Flik, and B. M. L. Verburg-van Kemenade: Interactions between the immune system and the hypothalamo-pituitary-interrenal axis in fish. Fish Shellfish Im munol., 9, 1-20, (1999). 15) C. H. Slater and C. B. Schreck: Testosterone alters the immune response of Chinook salmon, Oncorhynchus tshawytscha. Gen. Comp. Endocrinol., 89, (1993). 16) C. H. Slater, M. S. Fitzpatrick, and C. B. Schreck: Androgens and immunocompetence in salmonids: specific binding in and reduced immunocompetence of salmonid lymphocytes exposed to natural and synthetic androgens. Aquacult., 136, (1995). 17) C. H. Slater and C. B. Schreck: Physiological levels of testosterone kill salmonid leucocytes in vitro. Gen. Comp. Endocrinol., 106, (1997). 18) C. H. Slater, M. S. Fitzpatrick, and C. B. Schreck: Characteriza tion of an androgen receptor in salmonid lymphocytes: possible link to androgen-induced immunosuppression. Gen. Comp. En docrinol., 100, (1995). 19) C. H. Slater and C. B. Schreck: Season and physiological parameters modulate salmonid leucocyte androgen receptor affinity and abundance. Fish Shellfish Immunol., 8, (1998). 20) D. M. Jacobs and D. C. Morrison: Stimulation of a T-independent primary anti-hapten response in vitro by TNP-Iipopolysaccharide (TNP-LPS). J. Immunol., 114, (1975). 21) M. B. Rittenberg and A. A. Amkraut: Immunogenicity of trinitrophenyl-hemocyanin: production of primary and secondary anti-hapten precipitant. J. Immunol., 87, (1966). 22) J. S. Garvey, N. E. Creamer, and D. H. Sussdorf: DNP and TNPconjugeted proteins, in "Methods in Immunology, Third Ed.", W A. Benjamin, Massachusetts, 1977, pp ) A. F. Rowley: Collection, seperation and identification of fish leuco

6 Antibody Production and Steroids in Raibow Trout 855 cytes, in "Techniques in fish immunology" (ed. by J. S. Stolen, T. C. Fletcher, D. P. Anderson, B. S. Roberson, and W. B. van Muiswinkel), SOS, Fair Haven, New Jersey, 1990, pp ) A. D. Pickering and P. Christie: Changes in the concentrations of plasma cortisol and thyroxine during sexual materation of the hatch ery-reared brown trout, Salmo trutta L. Gen. Comp. Endocrinol., 44, (1981). 25) S. W. Lou, K. Aida, I. Hanyu, K. Sakai, M. Nomura, M. Tanaka, and S. Tazaki: Endocrine profiles in the females of a twice-annually spawning strain of rainbow trout. Aquaculture, 4, (1984). 26) S. W. Lou, K. Aida, 1. Hanyu, K. Sakai, M. Nomura, M. Tanaka, and S. Tazaki: Endocrine profiles in the females of a twice-annually spawning strain of rainbow trout. Gen. Comp. Endocrinol., 64, (1986). 27) R. A. Tripp, A. G. Maule, C. B. Schreck, and S. L. Kaattari: Cor tisol mediated suppression of salmoind lymphocyte response in vitro. Dev. Comp. Immunol., 11, (1987). 28) A. G. Maule, R. A. Tripp, S. L. Kaatari, and C. B. Schreck: Stress alters immune function and disease resistance in chinook salmon (Oncorhynchus tshawytscha). J. Endocrinol., 120, (1989). 29) M. C. Peleteiro and R. H. Richard: Identification of lymphocytes in the epidermis of the rainbow trout, Salmo gairdneri, Richardson. J. Fish Dis., 8, (1985). 30) M. C. Peleteiro and R. H. Richard: Immunocytochemical studies on immunoglobulin-containing cells in the epidermis of rainbow trout Salmo gairdneri, Richardson: influence of bath vaccination. J. Fish Biol., 32, , 1988.