Takashi SHIIBASHI * 1, Kouichi TAMAKI * 2, and Takaji IIDA * 2

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1 SUISANZOSHOKU 47(4), (1999) Oxygen-dependent Bactericidal Activity of Tilapia Neutrophils Takashi SHIIBASHI * 1, Kouichi TAMAKI * 2, and Takaji IIDA * 2 (Accepted October 1, 1999) Abstract: The involvement of superoxide (O2-), hydrogen peroxide (H2O2) and hypochiorous acid (HOCL) in oxygen-dependent bactericidal activity, together with their kinetics of oxygen metabolism during respiratory burst were investigated using tilapia, Oreochromis niloticus, neutrophils. In the presence of phorbol ester, respiratory burst was and oxygen radicals were generated in tilapia neutrophils. The rate of the oxygen consumption, O2- production and H2O2 production were 18.0 } 2. 73, 18.6 } 2.53 and 9.3 } 0.62 nmol/107 cells/min, respectively, showing a stoichiometrical proportion of 2: 2:1. This suggests that NADPH oxidase activity is responsible for respiratory burst and oxygen radical generation in tilapia neutrophils. The addition of a H2O2 scavenger, catalase, led to an apparent inhibition of bactericidal activity, whereas the addition of an O2 scavenger, superoxide dismutase, did not. Sodium azide (NaN3), an inhibitor of HOCL generating enzyme myeloperoxidase, apparently suppressed the bactericidal activity of tilapia neutrophils as well. These findings suggest that H2O2 and HOCL generated during respiratory burst are responsible for the oxygen-dependent bactericidal activity of tilapia neutrophils. Key words: Oreochromis niloticus; Neutrophil; Oxygen-dependent bactericidal activity; Respiratory burst Oxygen-dependent bactericidal activity is one of the important defense activities of neutrophils. When phagocytes ingest invading microorganisms, increased molecular oxygen uptake is observed, which is known as the respiratory burst1). This phenomenon is followed by produc tion of oxygen radicals that act as powerful bac vious study, we have demonstrated the universal existence of this component in fish neutrophils using the same antibody3). O2- is converted into more reactive secondary oxidant, hydrogen peroxide (H2O2), either by the spontaneous dismutation or in the presence of superoxide dismutase (SOD): tericidal agents. The superoxide anion (O2-), which is the first metabolite of the oxygen radi cal generation, is formed by the one electron re duction of molecular oxygen at the expense of NADPH: 2O2 + NADPH 2O2- + NADP+ + H+. In mammals, this reaction is catalyzed by NADPH oxidase. By immunoblotting using an anti-peptide antibody against the COON-terminal of cytochrome b558 large subunit corresponding synthetic peptide, the cytochrome b558 large subunit, an essential component of NADPH ox idase was detected in eel neutrophils2). In a pre 2O2- + 2H+ H2O2 + O2. The respiratory burst and the oxygen radical generation have been demonstrated in various fish phagocytes4). Itou et al.5) reported that the kinetics of oxygen metabolism during respiratory burst in Japanese eel, Anguilla japonica, neutrophils were similar to those in mammals. The importance of oxygen radicals in bactericidal activity of fish phagocytes have been described in rainbow trout, Oncorhynchus my kiss, macrophages6) and Japanese eel neutrophils7). These observations indicated that H2O2 is an important bactericidal agent. * 1 United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto , Kagoshima , Japan. * 2 Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-nishi 1-1, Miyazaki , Japan.

2 546 T. Shiibashi, K. Tamaki, and T. Iida While eel neutrophils possess little myeloperoxidase (MPO)8), it has been detected in neutrophils from most other fish species9). From the point of the oxygen-dependent bactericidal mechanism, the existence of this enzyme is meaningful, since in mammals the MPO catalyzes hypochlorous acid (HOCl) formation in the presence of H2O2 and chloride ion1) and HOC1 is thought to be the most active in phagocytic killing10). However, there are no reports about the generation of HOCl and its bactericidal activity in fish neutrophils. In the present study, we used tilapia, Oreochromis niloticus, neutrophils, which are MPO-positive, to investigat the involvement of 02-, H2O2 and HOCl in the oxygen-dependent bactericidal activity, together with their kinetics of oxygen Fish metabolism. Materials and Methods Tilapia (51.1 } 7.9 g body weight) hatched in our laboratory were kept at 25 C in tanks on a flow through water system and were fed daily on a dry pellet diet (Maruha, Japan). Isolation of Neutrophils Tilapia neutrophils were isolated using the swim bladder method described by Endo et al. 11) Collected neutrophils were washed twice with calcium, magnesium Hanks' balanced salt solution, and phenol red-free ph 7.4 (HBSS). The concentration of the neutrophils was adjusted to 5 X 106 or 1 X 107 cells/ml with HBSS and placed on ice until use. Kinetics of Oxygen Metabolisms of Tilapia Neutrothils during Resiratory Burst 02 production was measured by a ferricytochrome c reduction assay using a 96-well microtiter plate (Iwaki glass, Japan) according to Pick and Mizel12). The amount of 02 produced per well was calculated by the formula: nmol O2-/well = (absorbance at 550 nm X 100)/6.3. 1H2O2 production was measured by the scopoletin fluorescent method reported by Root et a1.13). The fluorescence from the scopoletin (excitation 360 nm, emission 460 nm) was measured with a Spectrafluoro Plus microplate fluorometer (TECAN, Austria) at 25 Ž. Simultaneously a standard curve was established using the concentrations of 0, 0. 1, 0. 2, 0. 5, 1.0 and 2.0,ƒÊM H2O2 in each experiment. Oxygen consumption was measured at 25 Ž using an oxygen monitor with a Clarktype oxygen electrode (YSI 5357 probe, Yellow Springs Instrument Co., U.S.A.) according to Itou et al. 5). The neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA; final concentration of 0.1ƒÊg/ml, Sigma, U. S. A.) in the assays. Chemiluminescent Assay The chemiluminescent (CL) assay was performed according to Iida and Wakabayashi14). In order to enhance CL, 3-aminophthal-hydrazide (1.4 mg/ml) (luminol; Sigma, U.S.A.) or 2-methyl- 6-phenyl-3, 7-dihydroimidazo[1,2-a]pyrazine-3-one (40 ƒêm) (CLA; Tokyo Kasei, Japan) was used as a probe. It has been reported that the luminolenhanced CL (L-CL) was dependent upon a MPO-mediated reaction, likely via generation of HOCl15). On the other hand, CLA, Cypridina luciferin analog, is highly specific and sensitive to 02-16). Sodium azide (NaN3; Wako, Japan) was used as an inhibitor of MPO, catalase (Sigma, U.S.A. ) or superoxide dismutase (SOD; Wako, Japan) was used as a scavenger of H2O2 or 02, respectively. The final concentrations of NaN3, catalase and SOD were 1 mm, 50 ƒêg/ml and 300 U/ml, respectively. Bacterial Strain Escherichia coli JAM 1239 was used as a target microorganism to measure the bactericidal activity of tilapia neutrophils. The bacteria were cultured on Triptosoya agar (TSA; Nissui, Japan) at 25 Žfor h and suspended with HBSS at a density of X 107 CFU/ml. Bactericidal Activity of Tilapia Neutrophils The bactericidal activity was measured according to the method of Secombes17) with some modifications. One hundred ƒêl of neutrophil suspension (5 ~ 106 cells/ml) was added to each well

3 Bactericidal Activity of Tilapia Neutrophils 547 Statistics % killing of bacteria= CFU after 60 or 120 min (1- CFU at 0 min ) ~ 100 The results were presented as a mean } standard error (S. E.) of the data from replicated examinations. Data was analyzed by the Student's t-test and significant differences were determined at p <0.05. of a 96 well tissue culture plate (Nunc, Denmark). The plate was then incubated for 30 min at 25 C to allow the cells to attach to the bottom of the plate. Bacterial suspension (100,al) was added to each well and the plate was shaken with the microplate mixer for 1 min. The plate was centrifuged at 150 ~ g for 5 min to let the bacteria contact with the cells. The wells were washed 6 times with HBSS to remove the free extracellular bacteria. The cells were then incubated at 25 Ž for 0, 60 and 120 min, respectively. After removing the supernatant, the cells were lysed by the addition of 200-ƒÊl ice-cold distilled water. Ten minutes later the contents in the wells were mixed by pipetting thoroughly and diluted in an appropriate amount of phosphate buffered saline (ph 7.4). Diluted bacterial suspension was spread onto TSA plates and were incubated for at least 24 h at 25 Ž. NaN3 (final concentration of 1 mm/ml) was added to the wells to block HOCI generation. Catalase (final concentration of 750,ƒÊg/ml) and SOD (final concentration of 1000 U/ml) were used to scavenge H2O2 and O2,-, respectively. Results were expressed as percent killing according to the formula: Results The Kinetics of Oxygen Metabolisms During Respiratory Burst The stimulation with PMA (0. l p g/ml) induced respiratory burst and oxygen radical generation in tilapia neutrophils. The amount of oxygen consumption, 02 - production and H2O2 production of the neutrophils were 18.0 } 2. 73, 18.6 } 2.53 and 9.3 } 0.62 nmol / 107cells / min, respectively (Table 1). Chemiluminescent Assay NaN3 partially inhibited L-CL (Fig. 1-a). The inhibitory effect of catalase on L-CL was equal Table 1. Kinetics * of oxygen metabolisms during respiratory burst of tilapia neutrophils * Mean } SE, (nmol/107 cells/min) (n = 5). Time after stimulation (min) Fig, 1. Effects of NaN3 and SOD on chemiluminescence of tilapia neutrophils. Arrows indicate the addition of PMA. a, Luminol-enhanced CL; b, CLA-enhanced CL. NaN3 or SOD (final concentration of 1 mm or 300 U/ml, respectively) was added to the reaction mixture. The results are representative from three individual experiments.

4 548 T. Shiibashi, K. Tamaki, and T. Iida to that of NaN3 (data not shown). By the addition of both NaN3 and SOD, L-CL was inhibited almost completely (Fig. 1-a). CLA-enhanced CL (CLA-CL) was not affected by NaN3 (Fig. 1-b), while CLA-CL was completely inhibited by the addition of SOD (Fig. 1-b). Catalase did not affect CLA-CL (data not shown). Bactericidal Activity of Tilapia Neutrophils Apparent killing activity by tilapia neutrophils was detected by using this modified bactericidal assay (Fig. 2). The mean values ( } SE) of % killing at 60 and 120 min were 55.3 } 5.1 and 66.1 } 6.0%, respectively. NaN3 decreased the the control (53.1 } 4.9%) (Fig. 4). On the other hand, the killing activity with SOD (51.9 }6.1%) was comparable to the control (54.7 } 4.9%) (Fig. 5). Discussion During the respiratory burst in tilapia neutrophils, oxygen consumption, 02- production and H2O2 production were detected at a ratio of 2:2:1 (Table 1). This observation was stoichiometrically similar to that found in Japanese eel5) and mammals, suggesting that NADPH oxidase activity is responsible for oxygen radical generation in tilapia neutrophils as killing activity to 39.5 } 2.8 %, showing a significant difference from the control (50.9 } 3.5%) (Fig. 3). Catalase caused a significantly reduced killing activity (34.2 } 5.0%) compared with Fig. 4. Effect of catalase on the bactericidal activity of tilapia neutrophils. Catalase (final concentration of 750 Đg/ml) was added to the test wells. NaN3 (final concentration of 1 mm) was added to both the control and the test wells to avoid the effect of MPOmediated system on bacterial killing. *, significant difference compared to the control (n=5). Fig. 2. Bactericidal activity of tilapia neutrophils (n=6). Fig. 3. Effect of NaN3 on the bactericidal activity of tilapia neutrophils. NaN3 (final concentration of 1 mm) was added to the test wells. *, significant difference compared to the control (n=5). Fig. 5. Effect of SOD on the bactericidal activity of tilapia neutrophils. SOD (final concentration of 1, 000 U/ml) was added to the test wells. Catalase (final concentration of 750,a g/ml) was added to both the control and the test wells to avoid the effect of H2O2 on bacterial killing (n=5).

5 Bactericidal Activity of Tilapia Neutrophils 549 well. This suggestion is consistent with the fact that cytochrome b558 large subunit, an essential component of NADPH oxidase, was detected in tilapia neutrophils by immunoblotting using an antipeptide antibody3) against the COOH-terminal of cytochrome b558 large subunit corresponding synthetic peptide. There were some combinations of animal species and bacterial species by which the oxygen radical generation was not induced18). By a preliminary investigation using NBT reduction method17), it was confirmed that E, coli TAM 1239 induced oxygen radical generation from tilapia neutrophils in the bactericidal assay used in the present study (data not shown). The addition of catalase led to an apparent inhibition of bactericidal activity, whereas the addition of SOD did not (Figs. 4 & 5), indicating that H2O2 is important for the bactericidal activity of tilapia neutrophils. These results agree with the report by Itou et al.7). They verified the significance of H2O2 in the oxygen-dependent bactericidal activity of Japanese eel neutrophils, because these neutrophils do not produce HOCI due to the lack of MP014). In the in vitro experiment, TAM 1239 used in the present E. coli study was completely killed by H2O2 at 5 mm within 60 min, and the median lethal concentration (LC50) at 60 min exposure was approximately 0.4 mm (data not shown). Since tilapia neutrophils produce about 9 nmol/107 cells/min of H2O2 (Table 1), its concentration in tilapia neutrophils would exceed the LC50 within 1 min and 5 mm in 5 min, according to the formula of Itou et al.7). This suggests that H2O2 produced by tilapia neutrophils likely contributes to the bactericidal activity. The inhibition of L-CL of tilapia neutrophils by the MPO inhibitor, NaN3 (1 mm), indicated that tilapia neutrophils showed HOCl generation via MPO-mediated reaction during the respiratory burst (Fig. 1-a), but NaN3 did not affected the 02- generation detected by CLA-CL at all (Fig. 1-b). From these results, 1 mm of NaN3 was used for the bactericidal assay to block only the HOCl killing. NaN3 apparently inhibited the bactericidal activity of tilapia neutrophils (Fig. 3), suggesting that the HOCl produced via MPOmediated reaction was responsible for the killing of E. coli. Because the quantity of HOCI generated during the respiratory burst of tilapia neutrophils was not determined in the present study, it is difficult to compare the effect of HOCl on the oxygen-dependent bactericidal activity to that of H2O2 precisely. The significance of HOCl on the bactericidal activity remains to be determined in fish neutrophils. The removal of H2O2 by catalase and the inhibition of generating HOCI by NaN3 did not suppress the bactericidal activity completely, suggesting that the tilapia neutrophils have oxygen-independent factors such as defensin, lysozyme or proteases. The bactericidal assay used in the present study may be suitable for estimation of the neutrophil bactericidal activity against opsonophagocytosisresistant bacteria such as Edwerdsiella tarda19~ and Aeromonas salmonicida20) because this assay makes neutrophils engulf materials without opsonin. Acknowledgements We express our sincere thanks to Drs.Makoto Endo, Terutoyo Yoshida, Faculty of Agriculture, Miyazaki University, for helpful discussion and Mr. Sam Harris for reviewing our manuscript. References 1) Edwards, S. W. (1994): Biochemistry and physiology of the neutrophils, in gthe Respiratory Burst: the Generation of Reactive Oxygen Metabolites and Their Role in Microbial Killing h, Cambridge University Press, New York, pp ) Itou, T., T. Iida, and H. Kawatsu (1998): Evidence for the existence of cytochrome b558 in fish neutrophils by polyclonal anti-peptide antibody. Dev. Comp. Im munol., 22, ) Shiibashi, T., T. Iida, and T. Itou (1999): Analysis of localization and function of the COOH-terminal corresponding site of cytochrome b558 in fish neutrophils. Dev. Comp. Immunol., 23, ) Secombes, C. J. and T. C. Fletcher (1992): The role of phagocytes in the protective mechanisms of fish. Ann. Rev. Fish Dis., 2, ) Itou, T., T. Iida, and H. Kawatsu (1996): Kinetics of oxygen metabolism during respiratory burst in Japanese eel neutrophils. Dev. Comp. Immunol., 20, ) Sharp, G. J. E. and C. J. Secombes (1993): The role of

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