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Enhancement of chemotactic activity of yellowtail (Seriola quinqueradiata) leucocytes by oral administration of quillaja saponin
Fish & Shellfish Immunology (1995) 5, 325–328
SHORT COMMUNICATION Enhancement of chemotactic activity of yellowtail (Seriola quinquerad iata) leucocytes by oral administration of quillaja saponin MANABU NINOMIYA*†, HAJIME HATTA†, MASARU FUJIKI†, MUJO KIM†, TAKEHIKO YAMAMOTO‡ AND RIICHI KUSUDA§ †Central Research Laboratories, Taiyo Kagaku Co. Ltd., Yokkaichi, Mie 510, Japan, ‡Department of Biotechnology, Fukuyama University, Fukuyama, Hiroshima 729-02, Japan and §Fish Disease Laboratory, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783, Japan (Received 17 January 1994, accepted in revised form 4 October 1994) Key words:
chemotactic activity, leucocyte, quillaja saponin, yellowtail.
It has been reported that immune responses in mice were activated by oral administration of quillaja saponin (QS), an extract of the soap tree, Quillaja saponaria (Mowat et al., 1991). Immunization with protein antigen in the presence of QS has also been reported to result in increased antibody production (Maharaj et al., 1986; Morein et al., 1987; Mowat et al., 1991; Kensil et al., 1991), delayed type hypersensitivity (Mowat et al., 1991), and promoted antigen-specific MHC class I restricted cytotoxic T lymphocytes (Takahashi et al., 1990). The action of QS has been attributed to its ability to modify cell membrane permeability and structure. In this regard, QS increases the permeability of the intestinal mucosa, resulting in increased uptake of viral antigens (Maharaj et al., 1986), and enhanced enteric uptake of human gamma globulin (HGG) in tilapia, Oreochromis mossambicus (Jenkins et al., 1991; 1992). It is well documented that chemotactic activity of phagocytes plays an important role in the host defense mechanisms (Handa et al., 1988). It is known that leucocytes from blood and organs of yellowtail, Seriola quinqueradiata, migrate quickly to the sites of inflammation (Hamaguchi et al., 1989), and motility of phagocytes is increased by serological factors and supernatants from stimulated lymphocyte cultures (Hamaguchi, 1991). Thus, it is of general interest to study the migration of leucocytes in response to the oral administration of QS in fish. This study reports the e#ect of oral administration of QS on the motility of yellowtail leucocytes in vitro. Yellowtail weighing 16–50 g were purchased from a commercial fish farm in Uranouchi Bay, Kochi Prefecture, Japan. Fish were kept in 800-l glass-fibre aquaria with circulating sea water at 21·8 to 24·5)C. QS was kindly provided by Maruzen Pharmaceuticals Co., Hiroshima, Japan. To determine the optimum dose of QS, 50, 5, 0·5 and 0 mg kg "1 body weight were administered. In the case of 50 mg kg "1 body weight administration, 250 mg of QS was made up to 100 ml with brown fish meal solution (brown fish meal powder:distilled water, 1:6, ww "1), QS content was 2·5 mg ml "1. This was orally intubated to the fish at 0·02 ml g "1 of fish body weight with a catheter. In the case of 5 and 0·5 mg kg "1 body weight administrations, 10-fold serial dilutions were intubated. As a control, brown fish meal solution alone was used. *Author to whom correspondence should be addressed. 325 1050–4648/95/040325+04 $08.00/0
? 1995 Academic Press Limited
M. NINOMIYA ET AL.
326
Table 1. The e#ect of oral administration of quillaja saponin (QS) to yellowtail leucocyte migration* Administration dose (mg kg "1 body weight) 50 5 0·5 0
Serum concentration in lower well (%)† 0·00 0·9‡ 2·2 0·5 1·2
(0·4)§ (0·9) (0·1) (0·4)
6·25 87·4 (22·1)Q 75·9 (11·3)¶ 21·8 (6·7) 21·0 (9·9)
*Migratory response of yellowtail leucocytes was measured 72 h after the oral intubation. †Zymozan-activated yellowtail serum. ‡Mean number of migrated leucocytes obtained from five fish per each treatment carried out in triplicate. §Figures in parenthesis indicate standard deviations. Significantly di#erent from 0 (mg kg "1 body weight), Q, P<0·05; ¶, P<0·01.
Five fish were used in each treatment. Seventy-two hours after QS administration, head kidney cell suspensions were prepared as described by Ninomiya et al. (1989) using Eagle’s minimum essential medium (Nissui Co., Tokyo, Japan) containing 10% fetal calf serum (MEM-10, pH 7·2, Wako Pure Chemical Industries Ltd., Osaka, Japan). The cells were counted in a haemocytometer and adjusted to 1#107 cells ml "1. The alternative complement pathway of normal yellowtail serum was activated by zymosan A (Sigma, St. Louis, MO) as described by Kodama et al. (1993). Chemotactic activity of head kidney cells was examined using Blind Well Chambers (Nucleopore Co., Pleasanton, CA). Zymosan-activated normal yellowtail serum was used as the chemotactic agent. Two hundred microlitres of MEM-10 alone or serum diluted with MEM-10 was added to the lower well of the Chambers. Nucleopore filters (5 ìm) were placed onto the wells. After fixing on the screws of the filter retainer, 0·2 ml of the head kidney cell suspension from sham or untreated control fish was added. Chambers were incubated at 25)C for 3 h. Thereafter, non-migrated cells on the surface of the filters were removed by washing with MEM-10. Filters were stained with Giemsa, and the cells which had migrated to the opposite side of the membrane were counted in 10 fields under a microscope. For the determination of activation period of chemotactic activity, 5 mg kg "1 body weight of QS was orally intubated once to fish, and the chemotactic activity was determined from five fish at 0, 24, 48, 96, 192 and 384 h after QS administration. In order to determine whether the migration of leucocytes to activated serum was chemotactic or random migration (chemokinesis), a checkerboard assay was performed using the method of Kodama et al. (1993). In certain experiments, 5 mg kg "1 body weight of QS was orally intubated once to three fish and the chemotactic activity was determined at 48 h after QS administration. Zymosan-activated yellowtail serum was added to concentrations of 6·25, 1·56 and 0% both into the upper and lower wells and the subsequent leucocyte migration was assessed as described above. Statistical analysis was performed by Student’s t-test. The migratory response of yellowtail leucocytes following the oral administration of QS is shown in Table 1. The migratory response of leucocytes was not increased by administration of 0·5 mg of QS kg "1 body weight, but it was significantly increased by administration of 5 (P<0·01) and 50 mg kg "1 body weight (P<0·05). The migratory response of leucocytes was monitored for a period of 24 to 384 h after QS administration, and the response was significantly increased at 48 h (P<0·01) and 96 h (P<0·05) (Fig. 1). As shown in Table 1 and Fig. 1, the observed chemotactic activity in the absence of both QS and serum at the onset of the experiment in Fig. 1 and equivalent activity in Table 1 were di#erent, presumable as the water temperature and body size of fish were di#erent in each set of experiments. The result of the checkerboard assay
CHEMOTACTIC ACTIVITY OF SERIOLA QUINQUERADIATA LEUCOCYTES
327
Fig. 1. Activation period of migratory response of yellowtail leucocytes 0, 24, 48, 96, 192 and 384 h after oral administration of 5 mg kg "1 body weight of quillaja saponin (QS). The migratory response was measured in the presence of zymosan-activated yellowtail serum (-, 6·25%; ,, 1·56%; ., 0%). Statistical significance (*, P<0·01; **, P<0·05) compared to 0 h. Bars show standard deviations from five fish, and the experiment was carried out in triplicate.
Table 2. Checkerboard assay of migratory response of yellowtail leucocytes* Serum concentration in upper well† (%) 0·00 1·56 6·25
Serum concentration in lower well (%) 0·00
1·56
6·25
2·6‡ (2, 4, 2) 2·6 (6, 1, 1) 2·0 (3, 2, 0)
18·3 (20, 15, 20) 10·3 (17, 8, 6) 5·3 (7, 8, 1)
35·3 (19, 38, 49) 19·3 (16, 23, 19) 12·7 (8, 18, 12)
*Migratory response of yellowtail leucocytes was measured 48 h after oral administration of quillaja saponin. †Zymosan-activated yellowtail serum. ‡Mean number of migrated leucocytes obtained from three fish.
of migratory response of leucocytes is shown in Table 2. When the serum concentrations on both sides of the membrane were equal (0, 1·56 and 6·25%), the responses were 2·6, 10·3 and 12·7, respectively. Thus, the increase of migratory response was proportional to the increase of serum concentrations, indicating the degree of chemokinesis. However, the response was much greater when the gradient of serum concentration increased (0% in upper and 6·25% in lower well), thus demonstrating that chemotaxis was dominating the response. This result argues that leucocytes from QS-treated fish are more sensitive to the complement gradient than untreated fish.
328
M. NINOMIYA ET AL.
Chemotactic activity of yellowtail leucocytes towards pathogens has been reported to be induced by stimulated lymphocyte supernatants containing lymphokines (Hamaguchi, 1991). As for rainbow trout, Oncorhyncus mykiss, the chemotactic activity of phagocytes has been reported to be increased by the supernatant of phagocytes from fish injected with muramyl dipeptide (Kodama et al., 1993). To elucidate the mechanisms underlying the activation of phagocytes from fish by oral administration of QS, further study will be necessary.
References Hamaguchi, M., Tanaka, T. & Kusuda, R. (1989). Preparation of neutrophils and macrophages with peritoneal exudate cells in yellowtail (Seriola quinqueradiata). Nippon Suisan Gakkaishi 55, 1511–1515. Hamaguchi, M. (1991). Studies on cellular immune response of yellowtail Seriola quinqueradiata against pseudotuberculosis. Bulletin of Nansei National Fisheries Research Institute 24, 83–91. Handa, T., Mitsuyama, M., Serushago, B. A., Kuramori, K. & Nomoto, K. (1988). Co-operative e#ect of MCF and MAF (IFN-r) in the protection of mice against Listeria monocytogenes. Immunology 65, 427–432. Jenkins, P. G., Harris, J. E. & Pulsford, A. L. (1991). Enhanced enteric uptake of human gamma globulin by Quil-A saponin in Oreochromis mossambicus. Fish and Shellfish Immunology 1, 279–295. Jenkins, P. G., Harris, J. E. & Pulsford, A. L. (1992). Quantitative serological aspects of the enhanced enteric uptake of human gamma globulin by Quil-A saponin in Oreochromis mossambicus. Fish and Shellfish Immunology 2, 193–209. Kensil, C. R., Patel, U., Lennick, M. & Marciani, D. (1991). Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex. Journal of Immunology 146, 431–437. Kodama, H., Hirota, Y., Mukamoto, M., Baba, T. & Azuma, I. (1993). Activation of rainbow trout (Oncorhyncus mykiss) phagocytes by muramyl dipeptide. Developmental and Comparative Immunology 17, 129–140. Maharaj, I., Froh, K. J. & Campbell, J. B. (1986). Immune responses of mice to inactivated rabies vaccine administered orally: potentiation by quillaja saponin. Canadian Journal of Microbiology 32, 414–421. Morein, B., Lovgren, K., Hoglund, S. & Sundquist, B. (1987). The ISCOM: an immunostimulating complex. Immunology Today 8, 333–338. Mowat, A. M., Donachie, A. M., Reid, G. & Jarrett, O. (1991). Immune-stimulating complexes containing Quil A and protein antigen prime class I MHC-restricted T lymphocytes in vivo and are immunogenic by the oral route. Immunology 72, 317–322. Ninomiya, M., Muraoka, A. & Kusuda, R. (1989). E#ect of immersion vaccination of yellowtail by ribosomal vaccine prepared from Pasteurella piscicida. Nippon Suisan Gakkaishi 55, 1773–1776. Takahashi, H., Takeshita, T., Morein, B., Putney, S., Germain, R. N. & Berzofsky, A. (1990). Induction of CD8 + cytotoxic T cells by immunization with purified HIV-1 envelope protein in ISCOMs. Nature 344, 873–875.
SHORT COMMUNICATION Enhancement of chemotactic activity of yellowtail (Seriola quinquerad iata) leucocytes by oral administration of quillaja saponin MANABU NINOMIYA*†, HAJIME HATTA†, MASARU FUJIKI†, MUJO KIM†, TAKEHIKO YAMAMOTO‡ AND RIICHI KUSUDA§ †Central Research Laboratories, Taiyo Kagaku Co. Ltd., Yokkaichi, Mie 510, Japan, ‡Department of Biotechnology, Fukuyama University, Fukuyama, Hiroshima 729-02, Japan and §Fish Disease Laboratory, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783, Japan (Received 17 January 1994, accepted in revised form 4 October 1994) Key words:
chemotactic activity, leucocyte, quillaja saponin, yellowtail.
It has been reported that immune responses in mice were activated by oral administration of quillaja saponin (QS), an extract of the soap tree, Quillaja saponaria (Mowat et al., 1991). Immunization with protein antigen in the presence of QS has also been reported to result in increased antibody production (Maharaj et al., 1986; Morein et al., 1987; Mowat et al., 1991; Kensil et al., 1991), delayed type hypersensitivity (Mowat et al., 1991), and promoted antigen-specific MHC class I restricted cytotoxic T lymphocytes (Takahashi et al., 1990). The action of QS has been attributed to its ability to modify cell membrane permeability and structure. In this regard, QS increases the permeability of the intestinal mucosa, resulting in increased uptake of viral antigens (Maharaj et al., 1986), and enhanced enteric uptake of human gamma globulin (HGG) in tilapia, Oreochromis mossambicus (Jenkins et al., 1991; 1992). It is well documented that chemotactic activity of phagocytes plays an important role in the host defense mechanisms (Handa et al., 1988). It is known that leucocytes from blood and organs of yellowtail, Seriola quinqueradiata, migrate quickly to the sites of inflammation (Hamaguchi et al., 1989), and motility of phagocytes is increased by serological factors and supernatants from stimulated lymphocyte cultures (Hamaguchi, 1991). Thus, it is of general interest to study the migration of leucocytes in response to the oral administration of QS in fish. This study reports the e#ect of oral administration of QS on the motility of yellowtail leucocytes in vitro. Yellowtail weighing 16–50 g were purchased from a commercial fish farm in Uranouchi Bay, Kochi Prefecture, Japan. Fish were kept in 800-l glass-fibre aquaria with circulating sea water at 21·8 to 24·5)C. QS was kindly provided by Maruzen Pharmaceuticals Co., Hiroshima, Japan. To determine the optimum dose of QS, 50, 5, 0·5 and 0 mg kg "1 body weight were administered. In the case of 50 mg kg "1 body weight administration, 250 mg of QS was made up to 100 ml with brown fish meal solution (brown fish meal powder:distilled water, 1:6, ww "1), QS content was 2·5 mg ml "1. This was orally intubated to the fish at 0·02 ml g "1 of fish body weight with a catheter. In the case of 5 and 0·5 mg kg "1 body weight administrations, 10-fold serial dilutions were intubated. As a control, brown fish meal solution alone was used. *Author to whom correspondence should be addressed. 325 1050–4648/95/040325+04 $08.00/0
? 1995 Academic Press Limited
M. NINOMIYA ET AL.
326
Table 1. The e#ect of oral administration of quillaja saponin (QS) to yellowtail leucocyte migration* Administration dose (mg kg "1 body weight) 50 5 0·5 0
Serum concentration in lower well (%)† 0·00 0·9‡ 2·2 0·5 1·2
(0·4)§ (0·9) (0·1) (0·4)
6·25 87·4 (22·1)Q 75·9 (11·3)¶ 21·8 (6·7) 21·0 (9·9)
*Migratory response of yellowtail leucocytes was measured 72 h after the oral intubation. †Zymozan-activated yellowtail serum. ‡Mean number of migrated leucocytes obtained from five fish per each treatment carried out in triplicate. §Figures in parenthesis indicate standard deviations. Significantly di#erent from 0 (mg kg "1 body weight), Q, P<0·05; ¶, P<0·01.
Five fish were used in each treatment. Seventy-two hours after QS administration, head kidney cell suspensions were prepared as described by Ninomiya et al. (1989) using Eagle’s minimum essential medium (Nissui Co., Tokyo, Japan) containing 10% fetal calf serum (MEM-10, pH 7·2, Wako Pure Chemical Industries Ltd., Osaka, Japan). The cells were counted in a haemocytometer and adjusted to 1#107 cells ml "1. The alternative complement pathway of normal yellowtail serum was activated by zymosan A (Sigma, St. Louis, MO) as described by Kodama et al. (1993). Chemotactic activity of head kidney cells was examined using Blind Well Chambers (Nucleopore Co., Pleasanton, CA). Zymosan-activated normal yellowtail serum was used as the chemotactic agent. Two hundred microlitres of MEM-10 alone or serum diluted with MEM-10 was added to the lower well of the Chambers. Nucleopore filters (5 ìm) were placed onto the wells. After fixing on the screws of the filter retainer, 0·2 ml of the head kidney cell suspension from sham or untreated control fish was added. Chambers were incubated at 25)C for 3 h. Thereafter, non-migrated cells on the surface of the filters were removed by washing with MEM-10. Filters were stained with Giemsa, and the cells which had migrated to the opposite side of the membrane were counted in 10 fields under a microscope. For the determination of activation period of chemotactic activity, 5 mg kg "1 body weight of QS was orally intubated once to fish, and the chemotactic activity was determined from five fish at 0, 24, 48, 96, 192 and 384 h after QS administration. In order to determine whether the migration of leucocytes to activated serum was chemotactic or random migration (chemokinesis), a checkerboard assay was performed using the method of Kodama et al. (1993). In certain experiments, 5 mg kg "1 body weight of QS was orally intubated once to three fish and the chemotactic activity was determined at 48 h after QS administration. Zymosan-activated yellowtail serum was added to concentrations of 6·25, 1·56 and 0% both into the upper and lower wells and the subsequent leucocyte migration was assessed as described above. Statistical analysis was performed by Student’s t-test. The migratory response of yellowtail leucocytes following the oral administration of QS is shown in Table 1. The migratory response of leucocytes was not increased by administration of 0·5 mg of QS kg "1 body weight, but it was significantly increased by administration of 5 (P<0·01) and 50 mg kg "1 body weight (P<0·05). The migratory response of leucocytes was monitored for a period of 24 to 384 h after QS administration, and the response was significantly increased at 48 h (P<0·01) and 96 h (P<0·05) (Fig. 1). As shown in Table 1 and Fig. 1, the observed chemotactic activity in the absence of both QS and serum at the onset of the experiment in Fig. 1 and equivalent activity in Table 1 were di#erent, presumable as the water temperature and body size of fish were di#erent in each set of experiments. The result of the checkerboard assay
CHEMOTACTIC ACTIVITY OF SERIOLA QUINQUERADIATA LEUCOCYTES
327
Fig. 1. Activation period of migratory response of yellowtail leucocytes 0, 24, 48, 96, 192 and 384 h after oral administration of 5 mg kg "1 body weight of quillaja saponin (QS). The migratory response was measured in the presence of zymosan-activated yellowtail serum (-, 6·25%; ,, 1·56%; ., 0%). Statistical significance (*, P<0·01; **, P<0·05) compared to 0 h. Bars show standard deviations from five fish, and the experiment was carried out in triplicate.
Table 2. Checkerboard assay of migratory response of yellowtail leucocytes* Serum concentration in upper well† (%) 0·00 1·56 6·25
Serum concentration in lower well (%) 0·00
1·56
6·25
2·6‡ (2, 4, 2) 2·6 (6, 1, 1) 2·0 (3, 2, 0)
18·3 (20, 15, 20) 10·3 (17, 8, 6) 5·3 (7, 8, 1)
35·3 (19, 38, 49) 19·3 (16, 23, 19) 12·7 (8, 18, 12)
*Migratory response of yellowtail leucocytes was measured 48 h after oral administration of quillaja saponin. †Zymosan-activated yellowtail serum. ‡Mean number of migrated leucocytes obtained from three fish.
of migratory response of leucocytes is shown in Table 2. When the serum concentrations on both sides of the membrane were equal (0, 1·56 and 6·25%), the responses were 2·6, 10·3 and 12·7, respectively. Thus, the increase of migratory response was proportional to the increase of serum concentrations, indicating the degree of chemokinesis. However, the response was much greater when the gradient of serum concentration increased (0% in upper and 6·25% in lower well), thus demonstrating that chemotaxis was dominating the response. This result argues that leucocytes from QS-treated fish are more sensitive to the complement gradient than untreated fish.
328
M. NINOMIYA ET AL.
Chemotactic activity of yellowtail leucocytes towards pathogens has been reported to be induced by stimulated lymphocyte supernatants containing lymphokines (Hamaguchi, 1991). As for rainbow trout, Oncorhyncus mykiss, the chemotactic activity of phagocytes has been reported to be increased by the supernatant of phagocytes from fish injected with muramyl dipeptide (Kodama et al., 1993). To elucidate the mechanisms underlying the activation of phagocytes from fish by oral administration of QS, further study will be necessary.
References Hamaguchi, M., Tanaka, T. & Kusuda, R. (1989). Preparation of neutrophils and macrophages with peritoneal exudate cells in yellowtail (Seriola quinqueradiata). Nippon Suisan Gakkaishi 55, 1511–1515. Hamaguchi, M. (1991). Studies on cellular immune response of yellowtail Seriola quinqueradiata against pseudotuberculosis. Bulletin of Nansei National Fisheries Research Institute 24, 83–91. Handa, T., Mitsuyama, M., Serushago, B. A., Kuramori, K. & Nomoto, K. (1988). Co-operative e#ect of MCF and MAF (IFN-r) in the protection of mice against Listeria monocytogenes. Immunology 65, 427–432. Jenkins, P. G., Harris, J. E. & Pulsford, A. L. (1991). Enhanced enteric uptake of human gamma globulin by Quil-A saponin in Oreochromis mossambicus. Fish and Shellfish Immunology 1, 279–295. Jenkins, P. G., Harris, J. E. & Pulsford, A. L. (1992). Quantitative serological aspects of the enhanced enteric uptake of human gamma globulin by Quil-A saponin in Oreochromis mossambicus. Fish and Shellfish Immunology 2, 193–209. Kensil, C. R., Patel, U., Lennick, M. & Marciani, D. (1991). Separation and characterization of saponins with adjuvant activity from Quillaja saponaria Molina cortex. Journal of Immunology 146, 431–437. Kodama, H., Hirota, Y., Mukamoto, M., Baba, T. & Azuma, I. (1993). Activation of rainbow trout (Oncorhyncus mykiss) phagocytes by muramyl dipeptide. Developmental and Comparative Immunology 17, 129–140. Maharaj, I., Froh, K. J. & Campbell, J. B. (1986). Immune responses of mice to inactivated rabies vaccine administered orally: potentiation by quillaja saponin. Canadian Journal of Microbiology 32, 414–421. Morein, B., Lovgren, K., Hoglund, S. & Sundquist, B. (1987). The ISCOM: an immunostimulating complex. Immunology Today 8, 333–338. Mowat, A. M., Donachie, A. M., Reid, G. & Jarrett, O. (1991). Immune-stimulating complexes containing Quil A and protein antigen prime class I MHC-restricted T lymphocytes in vivo and are immunogenic by the oral route. Immunology 72, 317–322. Ninomiya, M., Muraoka, A. & Kusuda, R. (1989). E#ect of immersion vaccination of yellowtail by ribosomal vaccine prepared from Pasteurella piscicida. Nippon Suisan Gakkaishi 55, 1773–1776. Takahashi, H., Takeshita, T., Morein, B., Putney, S., Germain, R. N. & Berzofsky, A. (1990). Induction of CD8 + cytotoxic T cells by immunization with purified HIV-1 envelope protein in ISCOMs. Nature 344, 873–875.