Changes in Glycogen Content of Neutrophils in Eel, Anguilla japonica by Bacterial Infection

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1 Changes Glycogen Content Eel, Anguilla japonica Bacterial Infection Yoshiaki NAGAMURA* Hisatsugu WAKABAYASHI Department Fisheries, Faculty Agriculture, University Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan Glycogen content eel which jected some extraneous materials such as formal-killed cells, 2% solution case physiological sale determed ir locomotion phagocytosis also examed. eel jected bacteria had highest content neutrophil glycogen, ranged from 74.6 Đg to Đg per 107. values those jected 2% solution case physiological sale Đg about 45 Đg, respectively. In locomotion nuetrophils, from eel jected 2% solution case mi grated best, those from eel jected bacteria did next place. bacterial phagocyto sis examed vitro whole. Glycogen-rich showed most remarkable phagocytosis, though locomotion not promoted. Introduction Materials Methods are believed to play an important role defence agast bacterial fections. In mam mals are a major phagocytic cells appear rapidly at an flamatory site. In addition it is known that mammal clude a large amount glycogen ir cytoplasm ir glycogen are energy source for motility (KAKINUMA, 1976; WAGNER, 1947). We descrived that peri pheral eel jected bacteria showed changes PAS reaction se PAS positive substances iden tified as glycogen salivery digestion test (NAGAMURA WAKABAYASHI, 1983). In present study, we vestigated changes glycogen content eel jected various extraneous materials like bac teria, examed how glycogen is used as a source energy examg locomotion phagocytosis. * Present address: Medical Dept. Research Labora tories Ueno Fe Chemical Industries, Ltd., 1-127, Higashiarioka, Itami-shi, Hyogo, Japan. Experimental Fish Eel (Anguilla japonica, g body weight) cultured Fisheries Laboratory Tokyo University used. Extraneous Materials extraneous materials used formal killed cells Vibrio anguillarum - Edwardsiella tarda, 2% solution case as well as physiological sale as a control. Blood Samples Blood samples taken from eel which jected formal-killed cells bacteria., 2% solution case physiological sale be fore 12 hrs or 24 hrs. Most whole cells used for neutrophil isolation rest prepared for smears, which staed May-Grunwald Giemsa (MGG) Periodic acid Shiff reaction (PAS). Isolation separated from peripheral usg Ficoll-Metrizoate solution (Fig. 1). One ml undiluted carefully loaded onto Ficoll-Metrizoate solution as separation medium tube centrifuged for 30 m at

2 390 Yoshiuki NAOASUIRA Hisatsugu WAKAi.AYASHI maly pasteur pipette times density representation Ficoll-metrizoate used for cell a modification 3 showed to stance. After cubated at whole suspension regular E. removed hema-. whole cubated tarcla tervals 100 cubation, - usg cells sub staed a microscopy vitro filled chemotactic filter n phagocytosis neutro apparatus distance bacterial a millipore under examed as neutro- chamber this migration Phagocytosis lower measured filled. filter 1 hr. apparatus method millipore locomotion preparation, cubation, toxyl. After for serum 26 Ž determed chamber scheme chamber activated After three determed Boyden exame upper from hed suspension phil cells collected apparatus phils. erythrocytes. method. vitro 3 2 Montogomery's Figure separation. ir Locomotion discontuous content Locomotion Schematic Content Glycogen K RP. Glycogen Fig. 1. n shaked at smears staed cells. bacterial 26 C gently. prepared MGG at PAS. Fig. 2. separated metrizoate usg a Ficoll solution bacteria carefully phagocytized observed microscopically. Results Glycogen tent 3. Membrane filter apparatus. table, ranged from In eel whole layers centrifugation taed thrombocytes cells divided (Fig. 2). to lymphocytes. eel pg 60.1 jected glycogen to pg 2% glycogen Table content jected to pg shown glycogen jected fish lowest determation are 74.6 from control highest ranged 1500 rpm. this Fig. Content result per 107 case. about it. physiological content conshown bacteria, solution pg. As sale 45 it had pg. three I con- 2 Locomotiou migration distance eel

3 Glycogen Content Eel 391 Table 1. Glycogen content eel jected various irritants Fig. 4. Migration toward zymosan-activated serum. obtaed from eel which jected 2% solution case. jected formal-killed bacteria, 2% solution case physiological sale showed Fig. 4, Fig. 5 Fig. 6, respectively. eel jected 2% solution case showed a marked migration. In fish jected formal-killed bacteria, migrated considerably. In control fish jected phys iological sale, comparison eel jected 2% solution case or formal-killed bacteria, migration distance varied widely. Phagocytosis bacterial phagocytosis examed

4 392 Yoshiaki NAGAMURA Hisatsugu WAKABAYASHI Fig. 5. Migration toward zymosan-activated serum. obtaed from eel which jected formal-killed cells E. tarda. Fig. 6. Migration toward zymosan-activated serum. obtaed from eel which jected physiological sale. vitro usg whole cells eel jected formal-killed cells E. coli, 2% so lution case physiological sale. As shown Table 2, after cubation for 30 m, eel jected formal killed cells E. coli showed a marked phagocytosis - ir phagocytic rate 40% 56%. Whereas, phagocytic rate eel jected physiological sale 16% 17%. In case 2% solution case, it lowest. phagocytosis agast bac teria are shown Fig. 7. neutrophil phago cytized bacteria actively 30 m after cubation

5 Fig. 7. Phagocytosis neutrophil. 1: After cubation for 30 m. MGG sta. 2: After cubation for 30 m. PAS reaction. 3: After cubation for 12 hr. MGG sta. 4: After cubation for 12 hr. PAS reaction.

6 394 Yoshiaki NAGAMURA Hisatsgu WAKABAYASHI Table 2. Phagocytic activity leukocytes from eel agast E. tarda at 26 Ž (Fig. 7-1) cytoplasm ir staed well PAS (Fig. 7-2). After 12 hr cubation, ir had vacuoles, where phagocytized bacteria observed y became swollen, PAS reaction phagocytizg bacteria became less proment gradually (Fig. 7-3, 4). Discussion It is known that mammals conta a large amount glycogen ir cytoplasm (KAKINUMA, 1976; WAGNER, 1947). WAGNER (1947) reported that glycogen content white cells 4.23ƒÊg per 106 cells. In control fish eel, Glycogen content about 45ƒÊg per 107 cells, its value did not show clear defference comparison those mam mals. However, it is an terestg fact that eel jected bacterial cells have considerably larger amount glycogen cytoplasm than those eel jected 2% solution case or physiological sale. In locomotion, eel jected 2% solution case tend to migrate best next place those eel jected bacteria, followed control. In spite eel jected bacteria had a large amount glycogen, y migrated less actively than those from eel jected 2% solution case. refore, it is speculated that locomotion does not necessarily depend on a large amount glyco gen ir cells. Whereas, phagocytosis, eel jected formal-killed cells E. coli showed a marked phagocytosis after cubation for 30 m. From those fact, it is probable that glycogen is used as an energy source for phagocytosis rar than for ir locomotion. And it is to be noted that phagocytosis is likely to be taken place non specifically, from fact that - eel jected E. coil responded quickly for admistration E. tarda. Thus, play a major role primary response agast bacterial fections. References CATES, K. L., C. E. RAY, P. G. QUIE (1978): Modified boyden chamber method measurg polymorphonu clear leukocyte chemotaxis. In Leukocyte Chemo taxis, Raven Press, New York, pp KAKINUMA, K. (1976): Oxygen metabolism phagocytes. Cell, 12(4), MONTGOMERY, R. (1957): Determation Glycogen. Arch. Biochem. Biophys., 67, NAGAMURA, Y. H. WAKABAYASHI (1983): Periodic acid-shiff reaction Eel, Anguilla japonica. Fish pathology, 17(4), WAGNER, R. (1947): Studies on physiology white cell. Blood, 2,