Superoxide anion generation by pacific oyster (Crassostrea gigas) hemocytes: Identification by...

Comp. Biochem. Physiol. Vol. 105B, No. I, pp. 35-41, 1993 0305-0491/93 $6.00 + 0.00 Printed in Great Britain © 1993 Pergamon Press Ltd

SUPEROXIDE ANION GENERATION BY PACIFIC OYSTER (CRASSOSTREA GIGAS) HEMOCYTES: IDENTIFICATION

BY ELECTRON SPIN RESONANCE SPIN TRAPPING AND CHEMILUMINESCENCE ANALYSIS

KEISUKE TAKAHASHI,* TAKAAKI AKAIKE,~" KEIZO SATO,t KATSUYOSHI MORI*~ and HIROSHI MAEDA~'§

*Laboratory of Aquacultural Biology, Department of Applied Biosciences, Faculty of Agriculture, Tohoku University, Aoba-ku scndai 981, Japan; and ~'Department of Microbiology,

Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860, Japan (Tel. 81-96-344-2111, Ext. 6321; Fax 81-96-362-8362)

(Received 19 October 1992; accepted 18 November 1992)

Abstratq--l. The generation of a supcroxide anion (O~-) by hemocytes of the Pacific oyster, Crassostrea gigas, was investigated by using electron spin resonance spin trapping with the spin trap 5,5-dimethyl-l- pyrroline N-oxide (DMPO), and by the chemiluminescence method.

2. Oyster hemocytes released O[- on stimulation with phorbol myristate acetate as revealed by both methods.

3. A great difference was found in O[- -generating activity among three subpopulations of hemocytes, which were separated from each other by using Percoll density gradients.

4. Evidence obtained in our experiments suggests that a specific O[--forming system, which becomes functional upon adequate stimulation, exists in a specialized subpopulation of C. gigas hemocytes similar to the professional phagocytes in mammals.

INTRODUCTION

On stimulation by foreign particles or humoral sub- stances, mammalian phagocytes produce a superoxide anion radical (O~-), which is considered to be a defense-orientated molecule against intruding microorganisms (Root et al., 1975; Babior, 1984; Rossi, 1986). It has been clarified that respiratory burst oxidase (NADPH oxidase) in the mammalian system is the univalent reduction system that generates O[- from 02 with serial redox components,, i.e. NADPH, a flavoprotein and a low-potential cytochrome b, together with cytosolic proteins (Clark, 1990).

In previous studies (Anderson et al., 1973, Cheng, 1976), no appreciable production of active oxygen molecules related to the respiratory burst system was found in invertebrate hemocytes. In contrast, an investigation by Nakamura et al. (1985) demonstrated production of H202 by scallop amoebocytes in vitro. In addition, generation of O[- and/or H202 could be identified by chemiluminescence, photo- metric and colorimetdc methods in molluscan invert- ebrates (Dikkeboom et al., 1987; Adema et aL, 1991; Anderson et al., 1992; Pipe, 1992). However, details of the O[--forming system of invertebrate hemocytes (amoeboeytes) remain unclear.

Although it was found that hemocytes of the Pacific oyster exhibit strong phagocytic activity

~To whom reprint requests should be addressed. §To whom correspondence should be addressed.

35

against various foreign particles and microbes and have a potent microbicidal action (Takahashi et al., unpublished observation), there is no conclusive and precise evidence showing production of O~- by hemocytes of the Pacific oyster. We therefore investigated the generation of O~- by hemocytes of the Pacific oyster Crassostrea gigas by using various sensitive and specific methods for identification of oxygen free radicals, i.e. chemiluminescence using a newly devel- oped acridinium compound, and the electron spin resonance (ESR) spin trapping method.

MATERIALS AND METHODS

Chemicals

Both 5,5-dimethyl-l-pyrroline N-oxide (DMPO), of the highest purity (> 99%), used as a spin trap for E S R measurement, and phenyl-10-methyl-acridinium-9-carboxylate fluorosulfonate (PMAC), which has 10- to 100-fold higher sensitivity than the conventionally used acridinium salt lucigenin for detection of O~-, were synthesized by Dojindo Lab- oratories (Kumamoto, Japan). Cu, Zn-superoxide dismutase (SOD) from bovine erythrocytes, luminol, and cytochrome c (type VI) were purchased from Sigma Chemical Co. (St Louis, MO). Dimethyl sul- foxide (DMSO) and phorbol myristate acetate (PMA) were obtained from Nacalai Tesque Inc. (Kyoto, Japan). Diethylenetriamine-pentaacetic acid (DTPA) was purchased from Dojindo Laboratories. Catalase from bovine liver and xanthine oxidase from

36 KEISUgE TAKAHASH~ et al.

cow milk were from Boehringer-Mannheim GmbH (Mannheim, Germany). All other reagents were the highest analytical grade commercially available.

Animals

The Pacific oysters, C. gigas, with a shell length of 11-14 cm, were harvested from hanging cultures in Matsushima Bay, Miyagi, Japan, as described previously (Takahashi et al., 1986), and were used throughout this experiment.

Collection of hemocytes

Hemolymph was collected from the blood sinus of the adductor muscle by using plastic syringes. Hemo- cytes were obtained by centrifugation at 400g for 15 min at 4°C and were washed three times in bal- anced salt solution for marine molluscs (MMBSS; Machii and Wada, 1989). The cells suspended in MMBSS were kept on ice until used.

To separate the subpopulations of hemocytes, density gradient techniques with Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) were used. A discon-

tinuous gradient was prepared by overlaying 5 ml of Percoll solution at 1.030 g/ml density, 5 ml of Percoll at 1.045 g/ml density, 5 ml of Percoll at 1.065 g/ml density, and 5 ml Percoll at 1.080 g/ml density, from the top to the bottom (Cheng and Downs, 1988). The density gradient centrifugation was performed at 400g for 15 min at 4°C. After centrifugation, thr~e different subpopulations were clearly visible and were fractionated separately. The density of each subpopulation was determined by using the density-marker beads (Pharmacia) as described previously (Borre- gaard et al., 1983).

ESR spin trapping

O~- formed by oyster hemocytes was identified by using ESR spin trapping with DMPO. Cell suspensions (140/zl; final cell density of 5-8 × 106 cells/ml in MMBSS) were mixed with 0.1 #g/ml of PMA and 90mM DMPO in the presence of 500#M DTPA (final volume, 200/zl). The mixture was immediately transferred into the ESR quartz flat cell (inner size: 60 × 10 × 0.31 mm3; effective volume 160/zl). ESR

CA)

O 0 o o o 0 o o

a . - 1.4

D M ~ H ; ~ n 1.49 m T , ~ - 1.49 mT

MnO I

• o o

+

NnO |

1 mT Fig. 1. ESR spectra of DMPO spin adducts obtained with PMA-stimulated hemocytes of C. gigas. (A) The ESR spectrum was recorded at 3.5 rain after addition of PMA (final concentration, 0.1/~ g/ml) to the cell suspension (final density, 8 x 106 cells/ml) in the presence of 90raM DMPO and 500/~M DTPA in MMBSS. Signals marked with closed circles belongs to the adduct of superoxide anion radical (DMPO-OOH). Computer simulation of the experimental spectrum (A) is shown in (B), which consists of two components: DMPO-OOH (shown in C) and DMPO-OH (shown in D) by their hypcrfine splitting constants, as described in the figure. See text for details of ESR measurement and computer simulation.

Superoxide anion production by oyster 37

spectra were recorded on a JEOL JES-REIX ESR spectrometer (JEOL Ltd, Akishima, Japan) at room temperature, under the following conditions: modulation frequency, 100kHz; modulation amplitude, 0.079 mT; scanning field, 336.2 + 5 mT; response time, 0.3 sec; sweep time, 2 rain; microwave power, 40 mW; and microwave frequency, 9,421 GHz. Com- puter simulation for experimental spectra was performed with ESR Simulation Program ESS-20 (Labotec Co. Ltd, Tokyo, Japan), as described recently (Akaike et al.; 1992).

Chemiluminescence assay

The PMAC-evokcd chemiluminescence response induced by O~- generated from oyster hcmocytes was detected and quantitated by using a six-channel luminometer equipped with a data-analyzing computer system (Model LB 9505C, Laboratorium Bcrthold AG, Wildbed 1, Germany). The reaction was initiated by the addition of 50/zl of PMA (final concentration, 0.1 #g/ml) to 400/tl of hcmocytc suspension (final density, 2-6 x 106cells/ml in MMBSS) with 500/~M

(A) Oyster cell without PMA

(B)

(c)

(D)

(E)

(8) + Cu,Zn-SOD (150 U/ml)

. . . .

(B) + Cu,Zn-SOD (1 SO U/ral) + catalase (550 U/ml)

(F) (a) + D~SO (S~)

MnO MnO ; :

1 mT

Fig. 2. ESR spectra of DMPO spin adducts obtained with the oyster hemocyte suspension under various conditions. The spectra were recorded at 8.5 min after addition of PMA to the cell suspension. (A) Spectrum obtained for the cell suspension (5 × 106 cells/ml) without PMA. (B) Spectrum observed on stimulation with PMA (0.1 #g/ml). (C) Spectrum obtained after addition of PMA in the presence of SOD (150 U/ml). (D) Same as (C) but with ¢atalase (550 U/ml) instead of SOD. (E) Spectrum obtained with SOD and catalase. (F) Spectrum obtained in the presence of DMSO (S%). (G) Same as (F) but with 5%

ethanol instead of DMSO. The conditions of ESR measurement were same as those in Fig. 1.

3 8 K E I S U K E T A K A H A S H I et al.

1,50-

o 6 ~ .00 " I

,~

.N l-

- - ~ O.SO-

¢w

0 .00 ,'o z'o ;o ,'o s'o ~'o

Time after incubation (min)

Fig. 3. Time course of relative intensity of DMPO spin adduct signal with PMA-stimulated oyster hemocytes. Each ESR spectrum was obtained after various incubation periods of the hemocyte suspension (5 x 10 ~ cells/ml) in MMB$S with DMPO (90 mM) and DTPA (500/~M) after stimulation with PMA (0.1#g/ml). After recording the spectra, the relative signal intensity of DMPO-OH was determined by comparison with the standard signal intensity

of the manganese oxide (MnO). See text for details.

DTPA and 2 .4gM (0.1#g/ml) PMAC, and the chemiluminescence response was continuously recorded at 26°C during the 60-min incubation period.

Cytochrome c reduction assay

O~- produced by bemocytes was also tested by SOD-inhibitable cytochrome c reduction as described previously (Akaike et al., 1990). The reaction mixture for detection of O~- generated in hemocytes con- tained 80/~M cytochrome c and 2.5 x 105 cells/ml in

the presence of DTPA (500 g M). The cytochrome c reduction assay was started by adding 0.1 #g/ml PMA to bemocyte suspensions, and an increase in absorbance at 550-540 nm was recorded with a spec- trophotometer (Hitachi U-2000, Hitachi Instruments, Inc., Tokyo, Japan) in the presence or absence of SOD (100 U/ml) at room temperature. The optical density reading was converted to nanomoles of cytochrome c reduced by using the molar absorption coefficient of 19.1mM -1 cm -~ (Kakinuma and Minakami, 1978).

RESULTS AND DISCUSSION

The present study demonstrated the generation of O~- by hemocytes of C. gigas on simulation with PMA. ESR spin trapping showed that both DMPO- OOH and DMPO-OH adducts were produced by the hemocytes (Figs 1 and 2), as assigned by computer simulation of the experimental spectra. The DMPO- OOH signal generated by PMA-stimulated hemocytes rapidly changes to the DMPO-OH signal. Although O~- reacts with the spin trap DMPO to yield a DMPO-OOH (DMPO-O~-) signal, this radical adduct (DMPO-OOH) spontaneously decom- poses into DMPO-OH under biological conditions, as reported by Finkelstein et al. (1982) and Ueno et al. (1989). Therefore, formation of DMPO-OH does not necessarily mean that hydroxyl radical ('OH) has been trapped. A similar observation has been described by Oda et al. (1992).

To identify the radical species responsible for the generation of DMPO-OH, we examined the effects of SOD and catalase on the generation of DMPO-OH. Addition of SOD at 150 U/ml completely abolished production DMPO-OH, as shown in Fig. 2C. In addition, eatalase (550U/ml) had no appreciable

0.6

.-- 0 . 4 X

~ .

0.2

0.0 0

• , I

A

B

,D 40 SO 10 20 30

Time (rain)

Fig. 4. PMAC-cvoked chemiluminescence responses Of oyster hcmocytes on stimulation with PMA. Chemiluminescence evoked with PMAC by the PMA-stimulated hcmocytcs was measured by a multi- channel luminometer in the absence (A) or presence (B) of SOD (100 U/ml). (C) shows a luminol-dependent, but not PMAC-dependent, chemiluminescence response of PMA-stimulatcd hemocytes. Without PMA stimulation no appreciable photocounting was observed to be dependent on PMAC (D). All

measurements were done at 26°C. See text for details.


1,00 ~ 1.05 ~

1.0~ , ~ 0 20 30 40 50

Distance from bottom (mm)

Fig. 5. Density profile of discontinuous Percoll gradients. The discontinuous gradients were prepared with oyster cell suspension overlaid on the Percoll solutions; initial density, 1.030-1.080g/ml, from the top of the bottom. Arrows indicate visually determined positions of the three subpopulations of oyster hemocytes (subsets I, II and III). A linear standard curve for the density profile was obtained with density marker beads as shown in the figure. Columns indicate percent recovery of oyster hemocytes in each sub-

population.

effect on generation of the DMPO-OH signal. Simi- larly, generation of DMPO-OH was not inhibited by the addition of DMSO (5%) or ethanol (5%) (Fig. 2F and G). These results clearly indicate that O~- was generated in PMA-stimulated hemocytes of C. gigas and was trapped by DMPO to form DMPO- OOH, followed by decomposition to DMPO-OH. The relative signal intensity of DMPO-OH generated by the hemocytes remained high during the initial

60-min incubation period, and the maximal level was reached at 25 min after initiation of this reaction (Fig. 3).

Oyster hemocytes suspended in MMBSS were also subjected to analysis for the PMAC-evoked chemiluminescence response on stimulation with PMA. As shown in Fig. 4, PMA-stimulated hemocytes exhib- ited a strong chemiluminescence response in the presence of PMAC, whereas no appreciable response was observed without PMA stimulation. The photocounting increased rapidly within the 10-min incubation period, with the peak count of 0.65 x l06 cpm seen at 20 min after the addition of PMA to the cell suspension; the response then declined slowly. This time course of PMAC-evoked chemiluminescence response of stimulated hemocytes correlated well with that of DMPO-OH production by the cells (Fig. 3). About 70% of the luminescence response was inhibited in the presence of SOD (100 U/ml) in the assay mixture (Fig. 4B). In addition, we found that catalase (up to l l00U/ml) showed only 2.4% inhibition against the PMAC-evoked chemiluminescence response induced by PMA-stimulated oyster hemocytes (data not shown). Therefore, it seems that H202 is not a major molecule responsible for this chemiluminescence reaction. These results indicate that a large part of the PMAC-evoked chemiluminescence response reflects O;- generation by the oyster hemocytes.

Previously, Dikkeboom et al. reported O~- generation in molluscs by using the luminol-dependent chemiluminescence assay (Dikkeboom et al., 1987, 1988). We also tested the luminol-dependent luminescence response in oyster hemocytes. However, luminol was found to induce only a very weak chemiluminescence response by PMA-stimulated

I 0 .6

II

~ o.4

,~

(~0 .2 !

0.0 0 10 20 30 40 50 Time (min)

Fig. 6. PMAC-evoked chemiluminescence response of three different subpopulations of hemocytes. Subsets I, II and III were fractioned by discontinuous Percoll density gradients. Measurement of chemiluminescence was started by adding PMA (final concentration, 0.1 #g/ml) to the cell suspension of each subpopulation (final cell density, 5 x l0 s cell/ml) in MMBSS in the presence of PMAC (2.4 # M) and DTPA (500/~M). (A) shows the luminescence response of subset II; (B), that of subset I; and (C), that

of subset III. See text for details.

40 KE~StrKE TAKAHASm et al.

1.5

~ ~ 1.0 ~. ~ 0

~ ~ ~ E~ ~o~ .t~ ~"

~ "~ 0.$

&

, I , I , i 1 I , I I

° 'Co 30 ~0 90 ~ 0 ~so ~ 0

Time (rain)

Fig. 7. Time course of reduction of cyt~hrome c indued by oys~r hem~ytes. The reaction was initiated by addition o(PMA (0.1 ~g/ml) to the ~11 sus~nsion in MMBSS in the pre~n~ of cytochrome c (80 ~ M)

and DTPA (5~ ~M) in the absence (~) or p ~ n ~ (~) of SOD (1~ U/ml). See text for details.

hemocytes: 100-fold weaker than that of PMAC (Fig. 4A vs C). In a separate experiment, we com- pared the chemiluminescence efficacy of PMAC with other luminescence enhancers such as luminol, lucigenin and luciferin in the reaction with O~- in a system with xanthine and xanthine oxidase (Fridovich, 1970). The results showed that PMAC was 10- to 100-fold more sensitive than luminol, lucigenin and luciferine (unpublished observation). Thus, use of this novel luminescent compound, PMAC, seems to be beneficial for identifying O~- generation in biological systems other than oyster hemocytes, as revealed in our experiments.

In contrast, the inhibitory effect of SOD on the PMAC-evoked chemiluminescence response was not necessarily complete. This may be partly due to limited accessibility of SOD to the reaction sites of PMAC with O~-. More specifically, one might specu- late that PMAC, which is smaller in molecular size but more hydrophobic molecule than SOD, could interact with the membrane of the hemocytes or could be internalized more efficiently than the SOD molecule to the intracellular space, where O~- would possibly be formed, similar to the situation in mammalian phagocytes (Clark, 1990).

By using a Percoll discontinuous density gradient, oyster hemocytes were separated into three subpopulations. Quantitative determination of O~- generation assessed by PMAC-evoked chemiluminescence revealed that among the three subsets that were separated (Fig. 5), two subpopulations with higher (subset I) and medium (subset II) cell densities showed O~- production on stimulation with PMA (Fig. 6). The subpopulation with the lowest density (subset III) did not show the O~--generating activity. Microscopic observation of these cells (Giemsa staining) indicated that cells in subsets I and II were granulocytes, and

subset III was composed of hyalinocytes, based on characteristic features described by Cheng and Downs (1988). Therefore, oyster hemocytes could be differentiated into at least two distinct subpopulations according to O~--generating activity.

The present results obtained by ESR spin trapping and measurement of chemiluminescence provide definitive evidence of O~- production by hemocytes of the Pacific oyster, C. gigas, on stimulation with PMA. Furthermore, O1- generation by oyster hemocytes was substantiated by a cytochrome c reduction assay (Fig. 7), although 60-70% of cytochrome c reduction in the mixture of hemocytes was not inhibited by added SOD. This SOD-insensitive reduction of cytochrome c may be attributed to deficiencies in this assay procedure, i.e. nonspecific reduction by cytochrome c-reducing enzymes or agents.

It has been revealed that Pacific oyster hemocytes have strong phagocytic activity against various foreign particles and microbes and potent microbicidal action (Takahashi et al., unpublished). Therefore, it is suggested that the O[- produced by oyster hemocytes may be a defense-orientated molecule against microorganisms, as suggested previously by Nakamura et aL (1985) and Dikkeboom et aL (1988). It is also suggested that a specific O[--forming system, which becomes functional on adequate stimulation, is made operational in the specialized subpopulations of C. gigas hemocytes, similar to the professional phagocytes in mammals.

Further biochemical or molecular characterization of the oxygen radical-generating system of oyster hemocytes is now in progress in our laboratories.

Acknowledgements--We should like to thank Ms S. Ijiri for expert technical assistance and Ms Judith B. Gandy and Ms. R. Yoshimoto for editorial work and typing, respectively.


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Superoxide anion generation by pacific oyster (Crassostrea gigas) hemocytes: Identification by...

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