Developmental and Comparative Immunology,Vol. 16, pp. 287-294, 1992 Printed in the USA. All rights reserved.
0145-305X/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
PHAGOCYTOSIS AND HYDROGEN PEROXIDE PRODUCTION BY PHAGOCYTES OF THE SEA URCHIN Strongylocentrotu$ nudus Toshimitsu Ito,* Takeshige Matsutani,* Katsuyoshi Mori,* and Tadashi Nomurat *Department of Fishery Science, Faculty of Agriculture, Tohoku University, Sendai, Japan and tlwate Institute of Technology, Iwate Japan
(Submitted June 1991; Accepted September 1991)
DAbstract--Phagocytosis of erythrocytes by phagocytes from the sea urchin Strongylocentrotus nudus can occur in vitro, and is enhanced by opsonization with the coelomic fluid. This opsonic activity of coelomic fluid can be elevated over a 5-day period by injecting erythrocytes into the coelom. Phagocytes produce hydrogen peroxide during both resting and stimulated states. This result on hydrogen peroxide production is the first to be observed in echinoderms. During the stimulated state, phagocytes produce more hydrogen peroxide than resting phagocytes. However, hydrogen peroxide production by phagocytes is not affected by opsonic activity of the coelomic fluid. Phagocytes share similar functional properties with vertebrate macrophages and granulocytes.
[]Keywords - - Echinoderm; Phagocytosis; Opsonin; Hydrogen peroxide; Phagocyte;
Strongylocentrotus nudus. Introduction
Recently, a number of studies on the defense mechanisms of invertebrates have been reported from the standpoint of comparative immunology. In invertebrates, specific defense reactions by immunoglobulins have not been found, and there are many obscure points regarding recognition systems that react to foreign materials (1). In Echinodermata, hemolysins and hemagglutinins are defense Address correspondence to T. Ito, Department of Fishery Science, Faculty of Agriculture, Tohoku University, Aobaku Sendai 981, Japan.
factors related to recognition of foreign materials (1). Phagocytosis, which is a cellular defense reaction, and opsonic effect of coelomic fluid has also been observed in echinoid coelomocytes (2). There is little information on the mechanisms of opsonic effect and the disposal of phagocytosed foreign materials in echinoids. In mammalian phagocytes, phagocytosed materials are exposed to lysosomal enzymes or reactive oxygen intermediates. Among invertebrates, molluscan phagocytes produce reactive oxygen after they have been stimulated by foreign materials (3-7). However, the mechanism and the role of reactive oxygen production by invertebrate phagoc y t e s remains as yet unclear. The present study deals with opsonic effects of coelomic fluid and hydrogen peroxide production by phagocytes in the sea urchin, Strongylocentrotus nudus.
Materials and M e t h o d s
Sea Urchins The sea urchin, Strongylocentrotus nudus, was collected from the rocky shore in Onagawa Bay, Miyagi Prefecture, Japan. They were transferred to our laboratory and maintained in a recirculating seawater system at 15°C until use. The mean fresh weight and test d i a m e t e r w e r e 69.6 g and 53 mm, respectively.
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Chemicals and Reagents N-p-Tosyl-L-arginine methyl ester hydrochloride (TAME) was obtained from Aldrich Chem. Co (USA). Homovanillic acid (HVA) and horseradish peroxidase (HRP type II) were purchased from Sigma Chem. Co. (USA). Other reagents were obtained from Wako Pure Chemical Industries Ltd., Japan. HEPES (N-2-hydroxyethyipiperazine-N'-2-ethanesulfonic acid) buffered artificial s e a w at er (HASW; 460 mM N a C I , 9 . 4 m M K C 1, 4 8 . 3 m M MgC12 • 6HzO, 6.0 mM NaHCO 3, 10.8 mM CaC12 • 2H20, and 10 mM HEPES, pH 7.4), HzOz-assay reaction medium (RM; HASW containing 0.4 mM HVA, 4 U/mL HRP, and 2 mM NAN3), and glycine NaOH buffer (100 mM glycine, 100 mM NaCl and 25 mM EDTA, pH 12) were used after filtration with a sterile 0.45 txm pore size Millipore filter. Anticoagulant was prepared according to the method of Bertheussen and Seljelid (8) (HASW containing 3 mM caffeine, 50 mM 2 - m e r c a p t o e t h a n o l and 2 mM TAME).
Cell Culture Coelomic fluid containing coelomocytes was obtained using a syringe fitted with a 20-G needle by puncturing the peristominal membrane. A syringe was previously half filled with anticoagulant before use. The number of coelomocytes was counted using a Btirker-Tiirk hemocytometer under a phase contrast microscope. C o e l o m o c y t e s w er e c u l t i v a t e d in chamber slides (Nunc Inc., USA). To adhere coelomocytes onto chamber slides, 0.4 mL aliquots of coelomocyte suspensions were pipetted onto each chamber and preincubated for 10 min at 4°C. Then the culture was washed with 0.5 mL of HASW to remove nonadherent coelomocytes. The majority of adherent coelo-
T Ito et al.
mocytes were phagocytes. The chamber was refilled with HASW and used for observation of phagocytosis or for H~O2 assays.
Preparation of Fixed Erythrocytes Sheep and human red blood cells (ShRBC, HuRBC) were washed with Ca z+ and Mg 2+ free Hanks solution and fixed with 3% formaldehyde for 24 h. The e r y t h r o c y t e s were washed with HASW and resuspended in HASW prior to use.
Erythrocytes Injection and Opsonization Fixed erythrocytes (ShRBC, HuRBC) suspended in HASW (2 × 10 7 cells) were injected into the coelom. Animals injected with HASW alone were used for controls. Coelomic fluid was collected from sea urchins 1-7 days after injection and used for opsonization. Coelomic fluid was centrifuged at 11,300 g for 10 min at 4°C. The supernatant fluid was passed through a sterile 0.45 Ixm pore size Millipore filter. For opsonization of erythrocytes, fixed erythrocytes (1 x 108 cells) were incubated for 1 h at 4°C in 1.0 mL of coelomic fluid. After opsonization, the erythrocytes were washed with HASW and resuspended in HASW.
Observation of Phagocytosis Erythrocytes treated with coeiomic fluid or HASW were added to each phagocyte culture at a concentration of 1.0 x 107 cells/mL (~-15 erythrocytes per phagocyte). The cell culture was incubated for 30 min at 12°C. After incubation, the culture was washed in HASW and fixed with 2% glutaraldehyde for 1 h. Phagocytosis was examined under a light
Sea urchin opsonin and H202 production
microscope after staining with hematoxylin-eosin.
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Results
Opsonic Effect of Coelomic Fluid H202 Assay The quantitative assay of H202 was performed with a slight modification according to the methods of Nakamura et al. (3) and Takeshige et al. (9). C o e l o m o c y t e s were adhered to a chamber slide that had been filled with 0.65 mL of HASW and 0.25 mL of RM, and the culture was incubated for 30 min at 12°C after adding 0.1 mL of fixed erythrocytes or HASW. Then, 0.1 mL of glycine NaOH buffer was mixed to stop the oxidative reaction of HVA, and this mixture was centrifuged at 500 g for 10 min to remove cells and debris. The increase in fluorescence of cell-free supernatant was measured with a HITACHI 605-10S fluorescence spectrophotometer (excitation at 315 nm and emission at 425 nm). The fluorescence change was standardized by adding 0.1 mL of known amounts of H202 to the assay mixture (0.65 mL of HASW and 0.25 mL of RM). A 10 mM stock solution of H202 in distilled water was stored at 4°C in the dark. The actual concentration of this solution was determined from its absorption at 230 nm, using a molar absorption coefficient of 81 M-1 c m - l . H202 standard solutions were prepared by diluting the stock solution with HASW prior to use. The control experiments were performed with the following systems: 1. RM + phagocytes + HASW (without stimulant); 2. RM (without HRP) + phagocytes + stimulant; 3. RM (without HVA) + phagocytes + stimulant.
Statistical Analysis Data were compared using a paired student t-test. The level of significance was set at p < 0.05 and p < 0.01.
About 10% of phagocytes ingested ShRBC and HuRBC over a 30-min incubation period. Elongation of the incubation time led to an increase in the percentage of phagocytes. At the same time the percentage of phagocytes that ingested two or more RBCs increased. The percentage of phagocytes ingested erythrocytes (phagocytosis rate) against HuRBC treated with the coelomic fluid from noninjected sea urchins (CF-I) was higher than that against HuRBC treated with HASW (Table 1). F u r t h e r m o r e , the phagocytosis rate against erythrocytes treated with coelomic fluid from sea urchins injected with HuRBC (CF-III) or ShRBC (CF-IV) was higher than those of controls and CF-I. In particular, when ShRBC were treated with CF-IV, the phagocytosis rate was significantly higher than that of any other treatment. Pretreatment of erythrocytes with the coelomic fluid from sea urchins injected with RBCs tended to increase the percentage of phagocytic phagocytes that ingested two or more RBCs. These results indicate that opsonic activity exists in the coelomic fluid from S. nudus and is enhanced by injections of RBCs. The time-course changes in opsonic activity of the coelomic fluid from sea urchins injected with RBCs were examined. Phagocytosis rates against ShRBC treated with HASW were below 10% and were lower than those against ShRBC treated with any coelomic fluid through the experimental period (Fig. 1). In addition, phagocytosis rates against ShRBC treated with coelomic fluid from sea urchins injected with RBCs were significantly higher than those against ShRBC treated with coelomic fluid from sea urchins injected with HASW on the fifth day (Fig. 1). When HuRBC was treated with any coelomic fluid, phago-
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Table 1. Opsonic Effect of Coelomic Fluid from S. nudus Injected With Sheep or Human Erythrocytes One Day Before Observation of Phagocytosis In Vitro. % of Phagocytic
Phagocytes C o n t a i n i n g Type of
Pretreatment
Phagocytosis
RBC
of RBCs With
Rate (%)
1 RBC
2 RBC
~>3 RBC
ShRBC
HASW (control) CF-I CF-II CF-III CF-IV HASW (control) CF-I CF-II CF-III CF-IV
8.3 -+ 2.1 10.7 -+ 2.2 12.6 +- 1.3 15.1 -+ 1.5" 19.7 + 1.4t 8.8 -+ 1.4 13.9-+1.4" 15.8 +- 0.9* 16.0 -+ 0.5* 16.4 +- 2.0*
76 -+ 6 69 -+ 4 65 -+ 5 52 +- 5 64 -+ 1 77 -+ 5 72-+5 65 +- 6 38 -+ 3 50 -+ 4
17 -+ 7 22 -+ 6 20 -+ 2 25 -+ 2 18 -+ 2 14 _+ 6 20-+6 21 + 2 31 -+ 4 22 _+ 2
7 +- 3 9 -+ 4 14 -+ 6 23 -+ 6 18 + 2 9 -+ 5 8-+2 13 -+ 5 31 -+ 4 28 -- 2
HuRBC
Values are the mean -+ SE of four experiments. RBC: red blood cell (erythrocyte); ShRBC: sheep RBC; HuRBC: human RBC; HASW: HEPES artificial sea water; CF: coelomic fluid; CF-I: CF from noninjected urchin; CF-II: CF from urchin injected with HASW; CF-IIh CF from urchin injected with HuRBC; CF-IV: CF from urchin injected with ShRBC. * Significantly different from HASW (p < 0.05). t Significantly different from HASW, CF-I, CF-II, and CF-III (p < 0.05).
Production of HeOe by Phagocytes
c y t o s i s rates were similar to those against ShRBC and were higher than HASW treatment (Fig. 2). Opsonic activity of coelomic fluid from sea urchins injected with RBCs were also significantly higher than that of coelomic fluid from sea urchins injected with HASW on the fifth day (Fig. 2). Therefore, it was presumed that the time required to maximize opsonin activity in coelomic fluid by RBCs injection was 5 days.
No fluorescence was observed in the assay system without HRP or HVA. A linear relationship existed between the intensity of fluorescence of HVA and the amounts of H202 added to the assay system. H202 production by resting phagocytes was calculated at 1.95 nmoles H202/105 cells/30 min. On the other
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Days after injection Figure 1. C h a n g e s in rates of p h a g o c y t o s i s of ShRBC pretreated with the c o e l o m i c fluid f r o m S. n u d u s injected with erythrocytes. ShRBC were pretreated with HASW (O) or c o e l o m i c fluid f r o m S. n u d u s injected with ShRBC ( . ) , HuRBC (&), or HASW (rq). Each value shows the mean -+ SE of f o u r experiments.
Sea urchin opsonin and H202 production
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Days after injection Figure 2. Changes in rates of phagocytosis of HuRBC pretreated with the coelomic fluid from S. nudus injected with erythrocytes. HuRBC were pretreated with HASW (O) or coelomic fluid from S. nudus injected with ShRBC (11), HuRBC (&), or HASW (r]). Each value shows the mean +- SE of four experiments.
hand, phagocytes stimulated by ShRBC and H u R B C p r o d u c e d 2.34 nmoles H202/105 ceUs/30 min and 6.46 nmoles/ l05 cells/30 min, respectively (Table 2). There were great differences in the production of H202 among individuals. For example, the maximal production stimulated by HuRBC was about 50 times the minimum (data not shown), indicating the possibility that phagocytes had prev i o u s l y r e c e i v e d some stimulation. Hence, the effect of stimuli on H202 production by phagocytes was estimated as a value relative to the level of resting phagocytes. Time-course changes of H202 production by phagocytes, which were stimulated by RBCs treated with the coelomic fluid from sea urchins injected with RBCs, were examined. When phagoTable 2. In Vitro Production of H=O2 by S. nudu$ Phagocytes. Stimulant
H20 2 Production*
HASW (resting) ShRBC HuRBC
1.95 - 0.23 2.34 - 0.27 6.46 - 1.39t
Values are the mean - SE of 16 experiments. HASW: HEPES artificial sea water; ShRBC: sheep red blood cell; HuRBC: human red blood cell (type B). * Nanomoles H202/105 cells/30 min. 1 Significantly different from resting (p < 0.01).
cytes were stimulated by coelomic fluidtreated ShRBC, the relative H202 production was significantly lower than that of phagocytes stimulated by HASWtreated ShRBC on the first day (Fig. 3). On the second and third day, the relative H202 production by every group of phagocyte decreased. However, the relative H202 production by coelomic fluidtreated ShRBC stimulated phagocytes was more than that by HASW-treated ShRBC stimulated ones on the third day. Furthermore, on the fifth day, phagocytes stimulated by ShRBC that had been treated with coelomic fluid from sea urchins injected with HuRBC produced significantly higher amounts of H202 than phagocytes stimulated by ShRBC treated with other coelomic fluid or HASW. The same treatments were performed with HuRBC, and their stimulating effects on the H202 production activity by phagocytes were also examined (Fig. 4). Stimulating effects of HuRBC treated with coelomic fluid from sea urchins injected with RBCs tended to be higher than that of HASW-treated HuRBC on the third day. On the other days, stimulating effects of coelomic fluid-treated HuRBC were the same as or lower than that of HASW-treated HuRBC. In other cases, the relative H202 production by phagocytes stimulated by HASW-treated RBCs varied
292
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from day-to-day (Figs. 3 and 4). Perhaps the response capacity of phagocytes against RBCs varied with their condition.
ity. The echinoderm opsonin is thought to be a complement-like substance and not a lectin (2). Similarly, the opsonic activity of coelomic fluid from sea urchins injected with RBCs increased during a 5-day period. Thus, it is considered that injection of RBCs raised the opsonic activity. Such an inducement of internal defense factors has also been described in the sea cucumber Holothuria polii, whose coelomic fluid hemolysin levels rose over an 8-day period after injecting formalinized ShRBC (12). So it seems that the opsonin from S. nudus is a complement-like molecule. In contrast, echinoderm lectins were reported to have a sugar-binding specificity enabling them to discern between self and nonself (13). To determine the biochemical characteristics and function of the echinoderm opsonin, therefore, both hemolysin and hemagglutinin (lectin) should be separated from the coelomic fluid and examined for each opsonic activity. The echinoderm phagocytes, following ingestion of foreign materials, appear to migrate to the gills, respiratory trees, and/or axial organ, from where they may be discharged to the exterior (14). The extent of intracellular breakdown of phagocytosed materials is not clear. In the H. polii, its coelomocytes ingested formalinized ShRBC and digested them in phagocytic vacuoles (15). In addition,
Discussion I m m u n o g l o b u l i n and c o m p l e m e n t bind to foreign particles, and play an important role in their phagocytosis by vertebrate granulocytes, monocytes, and macrophages. Invertebrate sera, which lack immunoglobulins, have a range of factors that mediate lytic, agglutinating, and opsonic activities against various biological agents (1). Invertebrate opsonins are known as lectin or complementlike substances (2,10,11). The present study indicates the existence of a naturally occurring opsonin in the sea urchin S. nudus coelomic fluid. The same observation was already reported in S. d r o e b a c h i e n s i s c o e l o m i c fluid (2). ShRBC and HuRBC opsonized by coelomic fluid were ingested at a similar rate, indicating that the opsonin is able to bind both RBCs. Moreover, according to one conclusion, phagocytes have receptors for the opsonin and this system is employed to distinguish nonself from self. From the present study, however, it cannot be deduced whether or not the coeIomic fluid opsonin has binding specific250
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Days after injection Figure 3. Changes of in vitro H202production by phagocytes ingesting ShRBC that were pretreated with the coelomic fluid from S. nudus injected with erythrocytes. ShRBC were pretreated with HASW (©) or coelomic fluid from S. nudus injected with ShRBC (11), HuRBC (&), or HASW (CI). Each value shows the mean + SE of four experiments. *, H202 production (stimulated)/H202 production (resting) xlO0.
Sea urchin opsonin and H202 p r o d u c t i o n
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Figure 4. Changes of in vitro H202 production by phagocytes ingesting HuRBC that were pretreated with the coelomic fluid from S. nudus injected with erythrocytes. HuRBC were pretreated with HASW (0) or coelomic fluid from S. nudus injected with ShRBC ( ' 4 HuRBC (A), or HASW (r-I). Each value shows the mean -+ SE of four experiments. *, H202 production (stimulated)/H20 s production (resting) ×100.
the coelomocytes contained lysosomal enzymes, including acid and alkaline phosphatases, 13-glucuronidase, aminopeptidase, lipase, peroxidase, and serine proteases (16,17). Hence, it can be assumed that these lysosomal enzymes play a role in the intracellular breakdown of phagocytosed materials. Other biological defense factors that are released from echinoid coelomocytes have been r e f e r r e d to as e c h i n o c h r o m e - A . Echinochrome-A, which has bactericidal activity, was found in red spherule coelomocytes of the sea urchin Echinus esculentus (18,19). Since red spherule coelomocytes have no phagocytic activity (14), it c a n n o t be a s s u m e d t h a t echinochrome-A plays a role in the intracellular breakdown of phagocytosed materials. On the other hand, H. polii coelomocytes released hemolysin when coelomocytes were stimulated by ShRBC (12,20). This hemolysin possessed properties different from the naturally occurring hemolysin in the coelomic fluid and was localized only in amebocytes (20). It is therefore probable that hemolysin produced by amebocytes (phagocytic coelomocytes) recognizes and binds to nonself materials as an opsonin and acts together with lysosomal enzymes to dispose of phagocytosed materials. Mammalian leukocytes generate reac-
tive oxygen intermediates when they ingest foreign materials. These reactive forms of oxygen, superoxide anion, hydrogen peroxide, and hydroxyl radical, are well known to be implicated in biological defense mechanisms. For example, in mollusks, reactive oxygens are produced by hemocytes after they are stimulated by foreign materials (3-7). In the present study, we found that sea urchin phagocytes produce the reactive oxygen intermediate, hydrogen peroxide; this is the first report of this phenomenon in echinoderms. The phagocytic phagocytes produced more hydrogen peroxide than resting ones. The amount of hydrogen peroxide produced by phagocytes when these are stimulated by RBCs was different with respect to ShRBC and HuRBC. This indicates that S. nudus phagocytes recognize the difference between the surface properties of the two R B C s . Similarly, in the snail Biomphalaria glabrata, hemocytes recognize irradiated or fixed miracidia of Schistosoma mansoni but not untreated live miracidia (4). Accordingly, this implies that a specific recognition system may exist in S. nudus phagocytes. There was only a weak relation between the phagocytosis rate and the amount of hydrogen peroxide produced in S. nudus. These results suggest that the hydrogen
294
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peroxide production system activates a n o t h e r p a t h w a y for the opsonin recognition system. T h u s far t h e p r e s e n c e o f i n t e r n a l def e n s e f a c t o r s t h a t c a n be i n d u c e d b y foreign m a t e r i a l s has n o t b e e n c l a r i f i e d in echinoderms. However, perhaps echinod e r m s p o s s e s s an i n t e r n a l d e f e n s e f a c t o r r e l e a s e s y s t e m that can be a c t i v a t e d when they recognize invading foreign
m a t e r i a l s . T h e p r e s e n t r e s u l t s s e e m to s u p p o r t t h e v i e w t h a t sea u r c h i n p h a g o cytes share functional features with vert e b r a t e m a c r o p h a g e s an d g r a n u l o c y t e s .
Acknowledgement--This work was supported by a Grant-in-Aid (Bio Media Program) from the Ministry of Agriculture, Forestry, and Fisheries (BMP 92-IV-2-5).
References 1. Ratcliffe, N. A.; Rowley, A. E; Fitzgerald, S. W.; Rhodes, C. P. Invertebrate immunity: Basic concepts and recent advances. Intern. Rev. Cytol. 97:183-354; 1985. 2. Bertheussen, K. Complement-like activity in sea urchin coelomic fluid. Develop. Comp. Immunol. 7:21-31 ; 1983. 3. Nakamura, M.; Mori, K.; Inooka, S.; Nomura, T. In vitro production of hydrogen peroxide by the amoebocytes of the scallop Patinopecten yessoensis (JAY). Develop. Comp. Immunol. 9:407-417; 1985. 4. Shozawa, A. A reducing factor produced by haemocytes of Biomphalaria glabrata and its role on the host defence. Develop. Comp. Immunol. 10:636; 1986. 5. Dikkeboom, R.; Tijnagel, J. M. G. H.; Mulder, E. C.; Van der Knaap, W. P. W. Hemocytes of the pond snail Lymnaea stagnalis generate reactive forms of oxygen. J. Invertebr. Pathol. 49:321-331; 1987. 6. Dikkeboom, R. ; van der Knaap, W. E W. ; van den Bovenkamp, W.; Tijnagel, J. M. G. H.; Bayne, C. J. The production of toxic oxygen metabolites by hemocytes of different snail species. Develop. Comp. Immunol. 12:509520; 1988. 7. Adema, C. M.; van Deutekom-Mulder, E. C.; van der Knaap, W. E W.; Meuleman, E. A.; Sminia, T. Generation of oxygen radicals in hemocytes of the snail Lymnaea stagnalis in relation to the rate of phagocytosis. Develop. Comp. Immunol. 15:17-26; 1991. 8. Bertheussen, K.; Seljelid, R. Echinoid phagocytes in vitro. Exp. Cell. Res. 111:401-412; 1978. 9. Takeshige, K.; Matsumoto, T.; Shibata, R.; Minakami, S. Simple and rapid method for the diagnosis of chronic granulomatous disease, measuring hydrogen peroxide and superoxide anions released from leukocytes in whole blood. Clinica. Chimica. Acta. 92:329-335; 1979. 10. Renwrantz, L.; Stahmer, A. Opsonizing properties of an isolated hemolymph agglutinin and
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demonstration of lectin-like recognition molecules at the surface of hemocytes from Mytilus edulis. J. Comp. Physiol. 149:535-546; 1983. Fryer, S. E.; Hull, J. C.; Bayne, C. J. Phagocytosis of yeast by Biomphalaria glabrata: Carbohydrate specificity of hemocyte receptors and a plasma opsonin. Develop. Comp. Immunol. 13:9-16; 1989. Canicatti, C.; Parrinello, N. Hemagglutinin and hemolysin levels in the coelomic fluid from Holothuria polii (echinodermata) following sheep erythrocyte injection. Biol. Bull. 168:175-182; 1985. Blaese, R. M.; Fleisher, T. A.; Tosato, G.; Muchmore, A. V.; Greene, W. C. The role of cell surface lectin-carbohydrate interactions in cellular recognition, cooperation, and regulation. Am. J. Pediat. Hematol. Oncol. 5:199206; 1983. Smith, V. J. The echinoderms. In: Ratcliffe, N. A.; Rowley, A. E eds. Invertebrate blood cells, vol. 2. New York: Academic Press; 1981:513. Canicatti, C. ; D'Ancona, G. Cellular aspects of Holothuria polii immune response. J. Invertebr. Pathol. 53:152-158; 1989. Canicatti, C. Lysosomal enzyme pattern in Holothuria polii coelomocytes. J. Invertebr. Pathol. 56:70-74; 1990. Canicatti, C.; Tschopp, J. Holozyme A: one of the serine proteases of Holothuria polii coelomocytes. Comp. Biochem. Physiol. 96B:739742; 1990. Wardlaw, A. C.; Unkles, S. E. Bactericidalactivity of coelomic fluid from the sea urchin Echinus esculentus. J. Invertebr. Pathol. 32:25-34; 1978. Matthew, S.; Wardlaw, A. C. Echinochrome-A as a bactericidal substance in the coelomic fluid of Echinus esculentus (L.). Comp. Biochem. Physiol. 79B:161-165; 1984. Canicatti, C.; Ciulla, D.; Farinalipari, E. The hemolysin-producer coelomocytes in Holothuria polii. Develop. Comp. Immunol. 12:729736; 1988.
0145-305X/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
PHAGOCYTOSIS AND HYDROGEN PEROXIDE PRODUCTION BY PHAGOCYTES OF THE SEA URCHIN Strongylocentrotu$ nudus Toshimitsu Ito,* Takeshige Matsutani,* Katsuyoshi Mori,* and Tadashi Nomurat *Department of Fishery Science, Faculty of Agriculture, Tohoku University, Sendai, Japan and tlwate Institute of Technology, Iwate Japan
(Submitted June 1991; Accepted September 1991)
DAbstract--Phagocytosis of erythrocytes by phagocytes from the sea urchin Strongylocentrotus nudus can occur in vitro, and is enhanced by opsonization with the coelomic fluid. This opsonic activity of coelomic fluid can be elevated over a 5-day period by injecting erythrocytes into the coelom. Phagocytes produce hydrogen peroxide during both resting and stimulated states. This result on hydrogen peroxide production is the first to be observed in echinoderms. During the stimulated state, phagocytes produce more hydrogen peroxide than resting phagocytes. However, hydrogen peroxide production by phagocytes is not affected by opsonic activity of the coelomic fluid. Phagocytes share similar functional properties with vertebrate macrophages and granulocytes.
[]Keywords - - Echinoderm; Phagocytosis; Opsonin; Hydrogen peroxide; Phagocyte;
Strongylocentrotus nudus. Introduction
Recently, a number of studies on the defense mechanisms of invertebrates have been reported from the standpoint of comparative immunology. In invertebrates, specific defense reactions by immunoglobulins have not been found, and there are many obscure points regarding recognition systems that react to foreign materials (1). In Echinodermata, hemolysins and hemagglutinins are defense Address correspondence to T. Ito, Department of Fishery Science, Faculty of Agriculture, Tohoku University, Aobaku Sendai 981, Japan.
factors related to recognition of foreign materials (1). Phagocytosis, which is a cellular defense reaction, and opsonic effect of coelomic fluid has also been observed in echinoid coelomocytes (2). There is little information on the mechanisms of opsonic effect and the disposal of phagocytosed foreign materials in echinoids. In mammalian phagocytes, phagocytosed materials are exposed to lysosomal enzymes or reactive oxygen intermediates. Among invertebrates, molluscan phagocytes produce reactive oxygen after they have been stimulated by foreign materials (3-7). However, the mechanism and the role of reactive oxygen production by invertebrate phagoc y t e s remains as yet unclear. The present study deals with opsonic effects of coelomic fluid and hydrogen peroxide production by phagocytes in the sea urchin, Strongylocentrotus nudus.
Materials and M e t h o d s
Sea Urchins The sea urchin, Strongylocentrotus nudus, was collected from the rocky shore in Onagawa Bay, Miyagi Prefecture, Japan. They were transferred to our laboratory and maintained in a recirculating seawater system at 15°C until use. The mean fresh weight and test d i a m e t e r w e r e 69.6 g and 53 mm, respectively.
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Chemicals and Reagents N-p-Tosyl-L-arginine methyl ester hydrochloride (TAME) was obtained from Aldrich Chem. Co (USA). Homovanillic acid (HVA) and horseradish peroxidase (HRP type II) were purchased from Sigma Chem. Co. (USA). Other reagents were obtained from Wako Pure Chemical Industries Ltd., Japan. HEPES (N-2-hydroxyethyipiperazine-N'-2-ethanesulfonic acid) buffered artificial s e a w at er (HASW; 460 mM N a C I , 9 . 4 m M K C 1, 4 8 . 3 m M MgC12 • 6HzO, 6.0 mM NaHCO 3, 10.8 mM CaC12 • 2H20, and 10 mM HEPES, pH 7.4), HzOz-assay reaction medium (RM; HASW containing 0.4 mM HVA, 4 U/mL HRP, and 2 mM NAN3), and glycine NaOH buffer (100 mM glycine, 100 mM NaCl and 25 mM EDTA, pH 12) were used after filtration with a sterile 0.45 txm pore size Millipore filter. Anticoagulant was prepared according to the method of Bertheussen and Seljelid (8) (HASW containing 3 mM caffeine, 50 mM 2 - m e r c a p t o e t h a n o l and 2 mM TAME).
Cell Culture Coelomic fluid containing coelomocytes was obtained using a syringe fitted with a 20-G needle by puncturing the peristominal membrane. A syringe was previously half filled with anticoagulant before use. The number of coelomocytes was counted using a Btirker-Tiirk hemocytometer under a phase contrast microscope. C o e l o m o c y t e s w er e c u l t i v a t e d in chamber slides (Nunc Inc., USA). To adhere coelomocytes onto chamber slides, 0.4 mL aliquots of coelomocyte suspensions were pipetted onto each chamber and preincubated for 10 min at 4°C. Then the culture was washed with 0.5 mL of HASW to remove nonadherent coelomocytes. The majority of adherent coelo-
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mocytes were phagocytes. The chamber was refilled with HASW and used for observation of phagocytosis or for H~O2 assays.
Preparation of Fixed Erythrocytes Sheep and human red blood cells (ShRBC, HuRBC) were washed with Ca z+ and Mg 2+ free Hanks solution and fixed with 3% formaldehyde for 24 h. The e r y t h r o c y t e s were washed with HASW and resuspended in HASW prior to use.
Erythrocytes Injection and Opsonization Fixed erythrocytes (ShRBC, HuRBC) suspended in HASW (2 × 10 7 cells) were injected into the coelom. Animals injected with HASW alone were used for controls. Coelomic fluid was collected from sea urchins 1-7 days after injection and used for opsonization. Coelomic fluid was centrifuged at 11,300 g for 10 min at 4°C. The supernatant fluid was passed through a sterile 0.45 Ixm pore size Millipore filter. For opsonization of erythrocytes, fixed erythrocytes (1 x 108 cells) were incubated for 1 h at 4°C in 1.0 mL of coelomic fluid. After opsonization, the erythrocytes were washed with HASW and resuspended in HASW.
Observation of Phagocytosis Erythrocytes treated with coeiomic fluid or HASW were added to each phagocyte culture at a concentration of 1.0 x 107 cells/mL (~-15 erythrocytes per phagocyte). The cell culture was incubated for 30 min at 12°C. After incubation, the culture was washed in HASW and fixed with 2% glutaraldehyde for 1 h. Phagocytosis was examined under a light
Sea urchin opsonin and H202 production
microscope after staining with hematoxylin-eosin.
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Results
Opsonic Effect of Coelomic Fluid H202 Assay The quantitative assay of H202 was performed with a slight modification according to the methods of Nakamura et al. (3) and Takeshige et al. (9). C o e l o m o c y t e s were adhered to a chamber slide that had been filled with 0.65 mL of HASW and 0.25 mL of RM, and the culture was incubated for 30 min at 12°C after adding 0.1 mL of fixed erythrocytes or HASW. Then, 0.1 mL of glycine NaOH buffer was mixed to stop the oxidative reaction of HVA, and this mixture was centrifuged at 500 g for 10 min to remove cells and debris. The increase in fluorescence of cell-free supernatant was measured with a HITACHI 605-10S fluorescence spectrophotometer (excitation at 315 nm and emission at 425 nm). The fluorescence change was standardized by adding 0.1 mL of known amounts of H202 to the assay mixture (0.65 mL of HASW and 0.25 mL of RM). A 10 mM stock solution of H202 in distilled water was stored at 4°C in the dark. The actual concentration of this solution was determined from its absorption at 230 nm, using a molar absorption coefficient of 81 M-1 c m - l . H202 standard solutions were prepared by diluting the stock solution with HASW prior to use. The control experiments were performed with the following systems: 1. RM + phagocytes + HASW (without stimulant); 2. RM (without HRP) + phagocytes + stimulant; 3. RM (without HVA) + phagocytes + stimulant.
Statistical Analysis Data were compared using a paired student t-test. The level of significance was set at p < 0.05 and p < 0.01.
About 10% of phagocytes ingested ShRBC and HuRBC over a 30-min incubation period. Elongation of the incubation time led to an increase in the percentage of phagocytes. At the same time the percentage of phagocytes that ingested two or more RBCs increased. The percentage of phagocytes ingested erythrocytes (phagocytosis rate) against HuRBC treated with the coelomic fluid from noninjected sea urchins (CF-I) was higher than that against HuRBC treated with HASW (Table 1). F u r t h e r m o r e , the phagocytosis rate against erythrocytes treated with coelomic fluid from sea urchins injected with HuRBC (CF-III) or ShRBC (CF-IV) was higher than those of controls and CF-I. In particular, when ShRBC were treated with CF-IV, the phagocytosis rate was significantly higher than that of any other treatment. Pretreatment of erythrocytes with the coelomic fluid from sea urchins injected with RBCs tended to increase the percentage of phagocytic phagocytes that ingested two or more RBCs. These results indicate that opsonic activity exists in the coelomic fluid from S. nudus and is enhanced by injections of RBCs. The time-course changes in opsonic activity of the coelomic fluid from sea urchins injected with RBCs were examined. Phagocytosis rates against ShRBC treated with HASW were below 10% and were lower than those against ShRBC treated with any coelomic fluid through the experimental period (Fig. 1). In addition, phagocytosis rates against ShRBC treated with coelomic fluid from sea urchins injected with RBCs were significantly higher than those against ShRBC treated with coelomic fluid from sea urchins injected with HASW on the fifth day (Fig. 1). When HuRBC was treated with any coelomic fluid, phago-
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Table 1. Opsonic Effect of Coelomic Fluid from S. nudus Injected With Sheep or Human Erythrocytes One Day Before Observation of Phagocytosis In Vitro. % of Phagocytic
Phagocytes C o n t a i n i n g Type of
Pretreatment
Phagocytosis
RBC
of RBCs With
Rate (%)
1 RBC
2 RBC
~>3 RBC
ShRBC
HASW (control) CF-I CF-II CF-III CF-IV HASW (control) CF-I CF-II CF-III CF-IV
8.3 -+ 2.1 10.7 -+ 2.2 12.6 +- 1.3 15.1 -+ 1.5" 19.7 + 1.4t 8.8 -+ 1.4 13.9-+1.4" 15.8 +- 0.9* 16.0 -+ 0.5* 16.4 +- 2.0*
76 -+ 6 69 -+ 4 65 -+ 5 52 +- 5 64 -+ 1 77 -+ 5 72-+5 65 +- 6 38 -+ 3 50 -+ 4
17 -+ 7 22 -+ 6 20 -+ 2 25 -+ 2 18 -+ 2 14 _+ 6 20-+6 21 + 2 31 -+ 4 22 _+ 2
7 +- 3 9 -+ 4 14 -+ 6 23 -+ 6 18 + 2 9 -+ 5 8-+2 13 -+ 5 31 -+ 4 28 -- 2
HuRBC
Values are the mean -+ SE of four experiments. RBC: red blood cell (erythrocyte); ShRBC: sheep RBC; HuRBC: human RBC; HASW: HEPES artificial sea water; CF: coelomic fluid; CF-I: CF from noninjected urchin; CF-II: CF from urchin injected with HASW; CF-IIh CF from urchin injected with HuRBC; CF-IV: CF from urchin injected with ShRBC. * Significantly different from HASW (p < 0.05). t Significantly different from HASW, CF-I, CF-II, and CF-III (p < 0.05).
Production of HeOe by Phagocytes
c y t o s i s rates were similar to those against ShRBC and were higher than HASW treatment (Fig. 2). Opsonic activity of coelomic fluid from sea urchins injected with RBCs were also significantly higher than that of coelomic fluid from sea urchins injected with HASW on the fifth day (Fig. 2). Therefore, it was presumed that the time required to maximize opsonin activity in coelomic fluid by RBCs injection was 5 days.
No fluorescence was observed in the assay system without HRP or HVA. A linear relationship existed between the intensity of fluorescence of HVA and the amounts of H202 added to the assay system. H202 production by resting phagocytes was calculated at 1.95 nmoles H202/105 cells/30 min. On the other
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hand, phagocytes stimulated by ShRBC and H u R B C p r o d u c e d 2.34 nmoles H202/105 ceUs/30 min and 6.46 nmoles/ l05 cells/30 min, respectively (Table 2). There were great differences in the production of H202 among individuals. For example, the maximal production stimulated by HuRBC was about 50 times the minimum (data not shown), indicating the possibility that phagocytes had prev i o u s l y r e c e i v e d some stimulation. Hence, the effect of stimuli on H202 production by phagocytes was estimated as a value relative to the level of resting phagocytes. Time-course changes of H202 production by phagocytes, which were stimulated by RBCs treated with the coelomic fluid from sea urchins injected with RBCs, were examined. When phagoTable 2. In Vitro Production of H=O2 by S. nudu$ Phagocytes. Stimulant
H20 2 Production*
HASW (resting) ShRBC HuRBC
1.95 - 0.23 2.34 - 0.27 6.46 - 1.39t
Values are the mean - SE of 16 experiments. HASW: HEPES artificial sea water; ShRBC: sheep red blood cell; HuRBC: human red blood cell (type B). * Nanomoles H202/105 cells/30 min. 1 Significantly different from resting (p < 0.01).
cytes were stimulated by coelomic fluidtreated ShRBC, the relative H202 production was significantly lower than that of phagocytes stimulated by HASWtreated ShRBC on the first day (Fig. 3). On the second and third day, the relative H202 production by every group of phagocyte decreased. However, the relative H202 production by coelomic fluidtreated ShRBC stimulated phagocytes was more than that by HASW-treated ShRBC stimulated ones on the third day. Furthermore, on the fifth day, phagocytes stimulated by ShRBC that had been treated with coelomic fluid from sea urchins injected with HuRBC produced significantly higher amounts of H202 than phagocytes stimulated by ShRBC treated with other coelomic fluid or HASW. The same treatments were performed with HuRBC, and their stimulating effects on the H202 production activity by phagocytes were also examined (Fig. 4). Stimulating effects of HuRBC treated with coelomic fluid from sea urchins injected with RBCs tended to be higher than that of HASW-treated HuRBC on the third day. On the other days, stimulating effects of coelomic fluid-treated HuRBC were the same as or lower than that of HASW-treated HuRBC. In other cases, the relative H202 production by phagocytes stimulated by HASW-treated RBCs varied
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from day-to-day (Figs. 3 and 4). Perhaps the response capacity of phagocytes against RBCs varied with their condition.
ity. The echinoderm opsonin is thought to be a complement-like substance and not a lectin (2). Similarly, the opsonic activity of coelomic fluid from sea urchins injected with RBCs increased during a 5-day period. Thus, it is considered that injection of RBCs raised the opsonic activity. Such an inducement of internal defense factors has also been described in the sea cucumber Holothuria polii, whose coelomic fluid hemolysin levels rose over an 8-day period after injecting formalinized ShRBC (12). So it seems that the opsonin from S. nudus is a complement-like molecule. In contrast, echinoderm lectins were reported to have a sugar-binding specificity enabling them to discern between self and nonself (13). To determine the biochemical characteristics and function of the echinoderm opsonin, therefore, both hemolysin and hemagglutinin (lectin) should be separated from the coelomic fluid and examined for each opsonic activity. The echinoderm phagocytes, following ingestion of foreign materials, appear to migrate to the gills, respiratory trees, and/or axial organ, from where they may be discharged to the exterior (14). The extent of intracellular breakdown of phagocytosed materials is not clear. In the H. polii, its coelomocytes ingested formalinized ShRBC and digested them in phagocytic vacuoles (15). In addition,
Discussion I m m u n o g l o b u l i n and c o m p l e m e n t bind to foreign particles, and play an important role in their phagocytosis by vertebrate granulocytes, monocytes, and macrophages. Invertebrate sera, which lack immunoglobulins, have a range of factors that mediate lytic, agglutinating, and opsonic activities against various biological agents (1). Invertebrate opsonins are known as lectin or complementlike substances (2,10,11). The present study indicates the existence of a naturally occurring opsonin in the sea urchin S. nudus coelomic fluid. The same observation was already reported in S. d r o e b a c h i e n s i s c o e l o m i c fluid (2). ShRBC and HuRBC opsonized by coelomic fluid were ingested at a similar rate, indicating that the opsonin is able to bind both RBCs. Moreover, according to one conclusion, phagocytes have receptors for the opsonin and this system is employed to distinguish nonself from self. From the present study, however, it cannot be deduced whether or not the coeIomic fluid opsonin has binding specific250
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Sea urchin opsonin and H202 p r o d u c t i o n
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Figure 4. Changes of in vitro H202 production by phagocytes ingesting HuRBC that were pretreated with the coelomic fluid from S. nudus injected with erythrocytes. HuRBC were pretreated with HASW (0) or coelomic fluid from S. nudus injected with ShRBC ( ' 4 HuRBC (A), or HASW (r-I). Each value shows the mean -+ SE of four experiments. *, H202 production (stimulated)/H20 s production (resting) ×100.
the coelomocytes contained lysosomal enzymes, including acid and alkaline phosphatases, 13-glucuronidase, aminopeptidase, lipase, peroxidase, and serine proteases (16,17). Hence, it can be assumed that these lysosomal enzymes play a role in the intracellular breakdown of phagocytosed materials. Other biological defense factors that are released from echinoid coelomocytes have been r e f e r r e d to as e c h i n o c h r o m e - A . Echinochrome-A, which has bactericidal activity, was found in red spherule coelomocytes of the sea urchin Echinus esculentus (18,19). Since red spherule coelomocytes have no phagocytic activity (14), it c a n n o t be a s s u m e d t h a t echinochrome-A plays a role in the intracellular breakdown of phagocytosed materials. On the other hand, H. polii coelomocytes released hemolysin when coelomocytes were stimulated by ShRBC (12,20). This hemolysin possessed properties different from the naturally occurring hemolysin in the coelomic fluid and was localized only in amebocytes (20). It is therefore probable that hemolysin produced by amebocytes (phagocytic coelomocytes) recognizes and binds to nonself materials as an opsonin and acts together with lysosomal enzymes to dispose of phagocytosed materials. Mammalian leukocytes generate reac-
tive oxygen intermediates when they ingest foreign materials. These reactive forms of oxygen, superoxide anion, hydrogen peroxide, and hydroxyl radical, are well known to be implicated in biological defense mechanisms. For example, in mollusks, reactive oxygens are produced by hemocytes after they are stimulated by foreign materials (3-7). In the present study, we found that sea urchin phagocytes produce the reactive oxygen intermediate, hydrogen peroxide; this is the first report of this phenomenon in echinoderms. The phagocytic phagocytes produced more hydrogen peroxide than resting ones. The amount of hydrogen peroxide produced by phagocytes when these are stimulated by RBCs was different with respect to ShRBC and HuRBC. This indicates that S. nudus phagocytes recognize the difference between the surface properties of the two R B C s . Similarly, in the snail Biomphalaria glabrata, hemocytes recognize irradiated or fixed miracidia of Schistosoma mansoni but not untreated live miracidia (4). Accordingly, this implies that a specific recognition system may exist in S. nudus phagocytes. There was only a weak relation between the phagocytosis rate and the amount of hydrogen peroxide produced in S. nudus. These results suggest that the hydrogen
294
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peroxide production system activates a n o t h e r p a t h w a y for the opsonin recognition system. T h u s far t h e p r e s e n c e o f i n t e r n a l def e n s e f a c t o r s t h a t c a n be i n d u c e d b y foreign m a t e r i a l s has n o t b e e n c l a r i f i e d in echinoderms. However, perhaps echinod e r m s p o s s e s s an i n t e r n a l d e f e n s e f a c t o r r e l e a s e s y s t e m that can be a c t i v a t e d when they recognize invading foreign
m a t e r i a l s . T h e p r e s e n t r e s u l t s s e e m to s u p p o r t t h e v i e w t h a t sea u r c h i n p h a g o cytes share functional features with vert e b r a t e m a c r o p h a g e s an d g r a n u l o c y t e s .
Acknowledgement--This work was supported by a Grant-in-Aid (Bio Media Program) from the Ministry of Agriculture, Forestry, and Fisheries (BMP 92-IV-2-5).
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