魚 病 研 究 Fish Pathology,32(2),121-125,1997.6

The Importance of Hydrogen Peroxide in Phagocytic Bactericidal

Activity of Japanese Eel Neutrophils

Takuya Itou*1, Takaji Iida*2 and Hiroshi Kawatsu*2

*1United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-21-24, Kagoshima 890, Japan

*2Department of Animal, Grassland and Fishery Sciences, Faculty of Agriculture, Miyazaki University, Gakuen Kibanadai-Nishi 1-1, Miyazaki 889-21, Japan

(Received December 25, 1996)

The bactericidal activity of Japanese eel neutrophils in the presence of oxygen radical scavengers was

investigated. Catalase inhibited bacterial killing, while superoxide dismutase and hydroxyl radical scavengers

(D-mannitol, sodium benzoate) did not. Superoxide dismutase had no synergetic effect with catalase. These observations indicate that hydrogen peroxide acts as a potent factor in the bactericidal process by eel neutrophils.

The phagocytic index of cytochalasin B-treated neutrophils was less than one third of that of control neutrophils,

although their phagocytic rate was more than 90%. Cytochalasin B, which inhibits phagosome formation without

suppressing radical production, decreased the bactericidal activity of the neutrophils, and catalase did not affect

the bacterial killing of cytochalasin B-treated neutrophils. Exposure to hydrogen peroxide at concentrations,

which are expected within eel neutrophils during the respiratory burst, effectively killed the bacteria tested. Thus,

it is suggested that the hydrogen peroxide-dependent bactericidal activity in eel neutrophils is expressed only

within phagosomes but not extracellularly and that phagosome formation is essential for this activity.

Key words: hydrogen peroxide, bactericidal activity, neutrophil, Japanese eel, cytochalasin B

Phagocytes engulf invading microorganisms and damage them with oxygen radicals. The production of

oxygen radicals follows an increase in oxygen consumption known as the respiratory burst (Babior, 1984). The

ability to generate oxygen radicals during the respiratory burst has been proved in a wide range of animal species,

even in hemocytes of invertebrates (Coen et al., 1991). Both respiratory burst and superoxide production were

demonstrated in Japanese eel neutrophils (Iida and Wakabayashi, 1995). During the respiratory burst in

eel neutrophils, oxygen consumption, superoxide production, and hydrogen peroxide production were detected

at a ratio of 2:2:1 (Itou et al., 1996) similar to that in mammals. It is well established in mammals that

oxygen radicals act as a powerful bactericidal agent against many bacteria (Baggiolini, 1984). In fish

phagocytes, Sharp and Secombes (1993) reported that rainbow trout macrophages demonstrate bactericidal

activity to Aeromonas salmonicida with reactive oxygen

species. Neutrophils are the first cells to be recruited to a site of infection and must respond quickly and

potently (Edwards, 1994). In spite of these importance, there is little study of fish neutrophils in oxygen

dependent bactericidal activity. In the present study, we investigated the role of oxygen radicals produced by eel neutrophils in the bactericidal process. Many

researchers have reported the importance of phagosome

formation for bacterial killing by mammalian phagocytes

(Zigmond and Hirsch, 1972; Densen and Mandell, 1978; Root et al., 1981). To further assess the contribution

of phagosome formation to bacterial killing by fish

phagocytes, we also examined the bactericidal activity of eel neutrophils treated with cytochalasin B, which

suppresses the phagosome formation of phagocytes.

Materials and Methods

Fish

Japanese eels (Anguilla japonica, mean weight 200g)

were purchased from a wholesaler and maintained at

25•Ž. The fish were not fed, and were used within two

weeks after purchase.

Isolation of Eel Neutrophils Neutrophils were isolated by the method reported by

Moritomo et al. (1988). Neutrophils were adjusted to

122 T. Itou, T. Iida and H. Kawatsu

2•~106 cells/ml with Hanks' balanced salt solution

(calcium, magnesium and phenol red-free HBSS; Nissui,

Japan).

Preparation of Bacteria We initially investigated the killing by eel neutrophils

of various bacteria such as Escherichia coli and Staphylococcus aureus which human phagocytes can kill effectively (Johnston et al., 1975). However, no highly sensitive species were found. Thus, we screened and selected a bacterium, designated as ESI, which is easily ingested and killed by eel neutrophils. ESI was isolated from the skin mucous of an apparently normal eel. ESI was classified as belonging to the family Pseudomona

daceae by characteristic tests, and cultured on Triptosoya

agar (Nissui, Japan) at 23•Ž. ESI were suspended with

HBSS at a density of 5-8•~106 CFU/ml after 24-36h

incubation.

Bactericidal Activity ESI suspensions (0.1ml) were opsonized with pooled

eel serum for 15min and then added to neutrophil suspensions (0.5ml). The total volume of the mixture was adjusted to 1 ml with HBSS (final serum concentration, 2.5%). Reaction mixtures with or without superoxide dismutase (SOD, superoxide scavenger, final concentration of 1000U/ml; wako, Japan) , catalase (hydrogen peroxide scavenger, final concentration of

Fig. 1. Effects of oxygen radical scavengers on killing of ESI by eel neutrophils . Bars indicate standard deviations from triplicate measurements. Neutrophils (1•~106 cells) were used in this experiments . The concentrations of SOD, catalase and D

-mannitol were 1000U/ml, 750ƒÊg/ml and 40mM, respectively . The results shown are representative of those of three

experiments.

Bacterial killing by H2O2 in eel neutrophils 123

750ƒÊg/ml; Sigma, USA), D-mannitol (hydroxyl radical

sacavenger, final concentration of 40mM; wako, Japan),

or sodium benzoate (hydroxyl radical scavenger, final

concentration of 20mM; wako, Japan) were prepared in

1.5ml tubes, which were then shaken at 12rpm at 25•Ž.

Fifty microliter aliquots of the mixtures were removed

after incubation for 0, 60 and 120min and suspended in 10

ml of distilled water to disrupt neutrophils. After

hypotonic lysis of neutrophils, 50ƒÊl aliquots of the

suspensions were spread on agar plates. The plates

were incubated for 12h at 37•Ž for colony counting.

The same experiments were also performed in the

presence of cytochalasin B (CB; Sigma, USA).

Neutrophil suspensions were preincubated with CB for

10 min before the addition of bacteria. The final

concentration of CB was 10ƒÊg/ml (in 1% dimethylsul

foxide).

Phagocytosis

Neutrophils (1•~106 cells) or CB-preincubated

neutrophils (1•~106 cells) and opsonized ESI (about 2.5•~10

8 CFU) in 1ml of HBSS containing 2.5% pooled eel

serum were shaken at 12 rpm at 25•Ž for 30min. After

incubation, 300ƒÊl aliquots of the mixtures were

suspended in 1ml of cold HBSS and appropriate dilutions

were smeared onto slide glasses by a centrifugal cell

collector (SC-2, Tomy, Japan). Phagocytosis was

observed under a light microscope following staining

with May-Grunwald Giemsa.

Results

Effects of Radical Scavengers on Bactericidal Activity ESI were killed effectively by eel neutrophils and

radical scavengers have no effect of bactericidal activity in the absence of eel neutrophils (Fig. 1). In the

presence of SOD (Fig. 1A) or hydroxyl radical scavengers (D-mannitol: Fig. 1C, sodium benzoate: data not shown), the efficiency of bacterial killing was not significantly different from that of controls. However, catalase markedly inhibited the bactericidal activity of

eel neutrophils (Fig. 1B). SOD had no synergetic effect

with catalase (Fig. 1B and D). Each assay was repeated three times and similar patterns were observed.

Effects of Cytochalasin B on Phagocytosis and Bactericidal Activity

The phagocytic index of CB-treated neutrophils was less than one third of that of controls, although their

phagocytic rate was more than 90% (Table 1). More than 95% of CB-treated neutrophils remained viable as determined by trypan blue dye exclusion. ESI showed a significant increase in survival rate in the bactericidal assay using CB-treated neutrophils (Fig. 2). Catalase did not enhance the inhibition of the killing induced by CB.

Killing of ESI by Exposure to Hydrogen Peroxide

To examine the toxicity of hydrogen peroxide against

ESI, the viability of ESI was measured after exposure to

various concentrations of hydrogen peroxide for 60min

(Fig. 3). Hydrogen peroxide at 500ƒÊM killed ESI

completely.

Discussion

Catalase inhibited the killing of ESI by eel neutrophils, whereas the other scavengers examined did not. These results indicated that hydrogen peroxide plays a major role in bacterial killing by eel neutrophils. Although the bactericidal activity was expected to increase due to the enhancement of hydrogen peroxide level by supplementation with SOD, the activity remained unchanged. We speculate that hydrogen peroxide derived from superoxide by spontaneous SOD

-independent dismutation had already reached sufficient level to kill bacteria. The killing of Aeromonas salmonicida by rainbow trout macrophages was also inhibited effectively by catalase (Sharp and Secombes, 1993). Their report suggested that hydroxyl radical is involved in the bactericidal activity since inhibition of the activity was observed in the presence of the hydroxyl

Table 1. Effect of cytochalasin B(CB) on phagocytosis of eel neutrophils

124 T. Itou, T. Iida and H. Kawatsu

Incubation time (min)

radical scavenger, formate and the possibility of

synergetic effect of SOD and catalase could not be

excluded. However, we did not find any effect of

hydroxyl radical on the killing of ESI . During

phagocytosis, hydrogen peroxide produced within

phagosomes can infiltration into bacteria, since it is both

stable and permeable (Baggiolini, 1984). Hydrogen

peroxide and Fe" have been suggested to generate

hydroxyl radical as a powerful oxidant within bacteria

(Imlay and Lim, 1988; Halliwell and Gutteridge, 1989).

This hydrogen peroxide to hydroxyl radical conversion

may have had a role in the present results. Interestingly,

Japanese eel neutrophils possess little myeloperoxidase

(MPO) (Park and Wakabayashi, 1989) which catalyzes

formation of hypochlorous acid from hydrogen peroxide

and chloride ions. This implies that hydrogen peroxide,

the substrate of MPO, ramains and is accumulated in

phagolysosomes of eel neutrophils due to MPO

deficiency. This increase in hydrogen peroxide level

may be advantageous for the bactericidal activity of eel

neutrophils.

CB inhibited phagocytosis and the killing of ESI by

eel neutrophils, suggesting that the formation of

phagosomes is important for the bactericidal process by

these cells, similarly to observations reported in

mammals (Zigmond and Hirsch, 1972; Densen and

Mandell, 1978; Root et al., 1981). CB did not suppress

superoxide production as determined by analysis of

CLA*-dependent chemiluminescence (data not shown) .

Catalase had no influence on the bactericidal activity in

the presence of CB. This fact also indicated that oxygen

radicals (hydrogen peroxide) kill microorganisms only

in phagosomes. Hydrogen peroxide at 500ƒÊM

completely killed ESI within 60min . Hydrogen

peroxide concentration (H2O2 conc.) at a specific time

point after commencement of H202 production can be

determined by the subsequent formula:

H2O2 conc. (uM)=p/vxt

p; H2O2 production (ƒÊM/min)

v; volume (l)

t; time (min)

If the volume of phagosomes is assumed to correspond

to that of whole eel neutrophils, it is calculated appro-

ximately 9mm3/10' neutrophils (average neutrophil

diameter: 12µm). We reported previously that eel

neutrophils produced about 5nM/107 cells/min of

hydrogen peroxide during the respiratory burst (Itou et

al., 1996). Under these conditions , H2O2 conc. in the

space containing bacteria would exceed 500ƒÊM in less

* CLA: Cypridina luciferin analog, 2-methyl-6-phenyl-3 , 7-dihydroimidazo [I, 2-a] pyrazine-3-one, is highly specific and sensitive

to superoxide.

Fig. 2. Effects of cytochalasin B on bactericidal activity by

eel neutrophils. The results represent the means •}

standard deviations from three experiments. Values

for cytochalasin B-treated neutrophils were

significantly different from those of controls (•¦:

p<0.05, * ; p<0.01) as determined by Student's t-test.

Fig. 3. Killing of ESI by exposure to hydrogen peroxide .

Viable number of ESI after 60min incubation in

hydrogen peroxide-free HBSS was determined as

100% survival.

Bacterial killing by H2O2 in eel neutrophils 125

than a few minutes. According to this hypothesis,

hydrogen peroxide produced by eel neutrophils could

have very effective bactericidal activity by itself.

The removal of oxygen radicals did not inhibit the

bactericidal activity completely. This suggested that

factors other than oxygen radicals, e.g. oxygen-indepen

dent ones such as defensins, lysozyme, proteases, etc.,

also contribute to bacterial inactivation in eel neutrophils.

These factors should be investigated in future studies.

Acknowledgments

We thank Drs. M. Endo and T. Yoshida, Department

of Animal, Grassland and Fishery Sciences, Faculty of

Agriculture, Miyazaki University, for invaluable sugges

tions. This work was supported in part by a Grant-in

-Aid for Scientific Research from the Ministry of Educa

tion, Science, Sports and Culture.

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