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Oxidant production by hemocytes of the eastern oyster, Crassostrea virginica (Gmelin)
IL5
.Iquorulrure. 107 (1992) 125-129 Elsevier Science Puhlisbers XV.. Amsterdam
Oxidant production by hemocytes ofthe eastern oystt-r, CV/lXXlSif2tiVl’Ygi;iica(GKl~l;n) Frank E. Fried1and Marvin R. Alvarez Deparlmen~
ofBiology,
L’niwrrrry
of.Sourh Florrda.
Tampa. FL, US4
ABSTRACT Friedl. F.E. and Alvarez M.R.. 1992. Oxidant productmn by hemocytes of the eaztem oyster, Crossostreo virginica (Gmelin). Aquacullure. 107: 125-129. In investigaIions of the oxygn metabolism of bemocytes of Ihe eastern oysrer. Crawslr~a virginicu. oxygen uptake was recorded from cell suspensions.Additionaily, hydrogen peroxide production by hemocyien was measured usicg a sensitive spectmfluorometric method. Additions of Concanavalin A or Zymosan significantly increased oxidant pmduciion over an endogenous level. whereas an increase with Pborbol Myristate Acetate was insignilicanl. Using a diaminobeniidine method, cytoplasmic deposits thought 10 be associawd with peroxidase activity were observed. The results suggest a hydropemxide metabolism capable of enhancing microbiocidal activities ofoyster phagocytes.
INTRODUCTION
Oyster hemolymph contains amoebocytic cells that are actively phagocytic. These multifunctional cells are generally considered to be important in nutrition and in the disposition ofabiotic and biotic materials. Foreign substances, depending on kind, may be killed, inactivated, digested, encapsulated, or carried out of the animal (diapedesis). With microorganisms, attachment and ingestion occur in a manner similar to that observed for many other animal types, hut the mechanisms of recognition and killing or inactivation may differ. With phagocytes of vertebrate animals, certain oxygen metaoolites are considered to be cytotoxic or cytocidal. These include the superoxide radical and
hydrogen peroxide. Furthermore, the effects of hydrogen peroxide are potentiated by hypohalides formed when a peroxidase and halide ions such as Cl-, Bf, and I- are present. A signature of this biocidal activity is a ‘respiratory burst'seen with phagocytosing mammalian ieukocytes and macrophages. Correspondence 10: F.E. Friedl. Departmeni Tampa, FL 3620.5150, USA.
0044-8486/9
2/%05.00
0 I992 Elwier
of Biotoiy, LIF 136, University
of South Florida,
Science Publishers B.V. All rights reserved.
126
FE.FRIEDLANDM.H.ALVAaEZ
Previous workers (Anderson et al., 1973 on insects; Cheng, 1976 on Mercenaria) did not find an increase in respiration associated with phagocytosis. On the other hand, other workers (Nakamura et al., 1985; Dikkeboom et al., 1987) have given evidence for the production of active oxygen metabolites by hemocytes of scallops and ;r,ails. In this communication we wish to report on evidence for a hydroperoxide r?etabolism ofpossible biocidal significance associated with the hemocytes of the eastern oyster, C’r?r~~~ea virpinica. METHODS AND RESULTS
Cell sources We obtained our hemocytes from oysters collected from Tampa Bay, Florida. The oysters were maintained in a recirculating artificial seawater system. Cells were obtained by needle-syringe aspiration from the pericardial cavity or an adductor muscle sinus. Cells from several animals were pooled as necessay to obtair! req~lred hemocyte concentrations. Oxvgen uptake by oyster hemocytes Oxygen uptake by piruled hemocyte preparations was recorded using a Clark-type oxygen electrode. Fig. 1 shows a digitized trace taken from a stirred sample of cells in artificial seawater at 27°C. An estimate of O2 uptake for that sample was 13 nmoles/lO min. The uptake of these small amounts of oxygen is reproducible and gives evidence of partial inhibition by cyanide and azide. Hydrogenperoxide productiorl WChave adapted the homovanillic acid method of Nakamura et al. ( 1985) for the measurement of hydrogen peroxide. These workers validated the use
121
OXIDANT PKDDVLTION BY HEMOWTES
of this sensitive fluorescence assay with the scallop Putinopecfen. Fig. 2 shows the results obtained from several combined experiments. Hemocytes (O.l1.0~ 1Q6 cells) were added to reactant mixtures (plus additives as noted). After I h at room temperature (23°C)) the reactions were terminated and the fluorescence of oxidized homovani!!ic acid was measured. Typically each experiment included a time-zero control, 2-3 incremental cell concentrations, and H,O, standards. An endogenous production of H,O, was appzent and the Kmskal-Wallis test (a nonparametric rank test) indicated a significant effect of additives on oxidant production. With the Wilcoxon signed ranks test, significantiy higher concentrations of oxidant were found upon ariditions of Concanavalin A (Con A) and Zymosan. The increase seen with
i
PMA
I-
0.800
1.000
n=6
10 9
a 7 5 5 4 3 2
1 ~ 00000
0.200
0.400
0.600
~e,,s per assay x 106 Fig. 3. H,O, production by ~)ysterhemocyw ! nnnomoles I-LO, vs. cell number-
Con A added).
128
F.E.FRIEDLAND MR. ALVAREZ
Phorbol Myristate Acetate (PMA) was insignificant however. Production of oxidant was found to be proportional to cell concentration (Fig. 3) and ancillary experiments indicated that catalase and oxygen deprivation reduce amounts of oxidant measured. Localization ofperoxidase Using a standard diaminobenzidine method, we found cytoplasmic deposits susesting the presence of peroxidase activity. These deposits, absent in controls, are spheroidal, resembling in shape the granules seen in certain hemocytes. CONCLUSIONS
We have shown that hemocytes of Cmsrorrreu virnlnrcu consume small amounts of oxygen that conceivably could be part of a hydroperoxide metabolism. We detected the endogenous production of an oxidant, presumably H,O,, and showed that more of it appeared wher cells were stimulated. Whether this hydrogen peroxide is associated with cytotoxic effects, an innoo uous consequence of altered cell membrane activity and/or leakage, or simply a normal pattern of oxygen metabolism is not known. Because of the avid
phagocytic activities of many of these cells, the presence of a peroxidase possibly able to potentiate the effects of H202 is of additional interest. We feel that our research strongly suggests the involvement of a hydroperoxide metabolism in biocidal activities of oyster hemocytes. ACKNOWLEDGEMENTS ‘*ve thank Ms. Jacqueline S. Johnson for her expert technical assistance with this research and are indebted to Ms. Carmen S. Patron for her work with hemocyte oxygen uptake. This article was developed under the auspices of
the Florida Sea Grant College Program with support from the National Oceanic and A’mospheric Administration, Oftice of Sea Grant, U.S. Departmeni of Commerce, Grant No. NA86AA-D-SG068. The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon.
REFERENCES
Anderson,R., Holmes,B. and Good, R.. 1973. Comparativebiochemistry of phagocytizing insect hemocytes. Camp.B&hem. Physiol.,46B:595-602. Cheng.T., 1976. Aspectsof substrueutdizationand energyrequirementdunna molluscan phagocytosis. J. Invert.Pathol.,27: 263-268.
OXIDANT
,‘RODUCTlON
BY HEMOWTES
129
Dikkeboom R.. Tijnagel, J., Molder. E. and Van der Knaap. W.. 1987.Hemocees of the pond snail Lymnceo rragnaY generatereactive form5 of oxygen.J. Invert. Pathol., 49: 321-331. Nakamura, M., Mori, K., Inooka, S. knd Nomura, T., 1985. Is vitrc protiction of hydrogen peroxide by the amoebocytesoftbc scallop,Parmoprcren~essoensis (Jay). Cev. Comp. Immunol., 9: 407-417.
.Iquorulrure. 107 (1992) 125-129 Elsevier Science Puhlisbers XV.. Amsterdam
Oxidant production by hemocytes ofthe eastern oystt-r, CV/lXXlSif2tiVl’Ygi;iica(GKl~l;n) Frank E. Fried1and Marvin R. Alvarez Deparlmen~
ofBiology,
L’niwrrrry
of.Sourh Florrda.
Tampa. FL, US4
ABSTRACT Friedl. F.E. and Alvarez M.R.. 1992. Oxidant productmn by hemocytes of the eaztem oyster, Crossostreo virginica (Gmelin). Aquacullure. 107: 125-129. In investigaIions of the oxygn metabolism of bemocytes of Ihe eastern oysrer. Crawslr~a virginicu. oxygen uptake was recorded from cell suspensions.Additionaily, hydrogen peroxide production by hemocyien was measured usicg a sensitive spectmfluorometric method. Additions of Concanavalin A or Zymosan significantly increased oxidant pmduciion over an endogenous level. whereas an increase with Pborbol Myristate Acetate was insignilicanl. Using a diaminobeniidine method, cytoplasmic deposits thought 10 be associawd with peroxidase activity were observed. The results suggest a hydropemxide metabolism capable of enhancing microbiocidal activities ofoyster phagocytes.
INTRODUCTION
Oyster hemolymph contains amoebocytic cells that are actively phagocytic. These multifunctional cells are generally considered to be important in nutrition and in the disposition ofabiotic and biotic materials. Foreign substances, depending on kind, may be killed, inactivated, digested, encapsulated, or carried out of the animal (diapedesis). With microorganisms, attachment and ingestion occur in a manner similar to that observed for many other animal types, hut the mechanisms of recognition and killing or inactivation may differ. With phagocytes of vertebrate animals, certain oxygen metaoolites are considered to be cytotoxic or cytocidal. These include the superoxide radical and
hydrogen peroxide. Furthermore, the effects of hydrogen peroxide are potentiated by hypohalides formed when a peroxidase and halide ions such as Cl-, Bf, and I- are present. A signature of this biocidal activity is a ‘respiratory burst'seen with phagocytosing mammalian ieukocytes and macrophages. Correspondence 10: F.E. Friedl. Departmeni Tampa, FL 3620.5150, USA.
0044-8486/9
2/%05.00
0 I992 Elwier
of Biotoiy, LIF 136, University
of South Florida,
Science Publishers B.V. All rights reserved.
126
FE.FRIEDLANDM.H.ALVAaEZ
Previous workers (Anderson et al., 1973 on insects; Cheng, 1976 on Mercenaria) did not find an increase in respiration associated with phagocytosis. On the other hand, other workers (Nakamura et al., 1985; Dikkeboom et al., 1987) have given evidence for the production of active oxygen metabolites by hemocytes of scallops and ;r,ails. In this communication we wish to report on evidence for a hydroperoxide r?etabolism ofpossible biocidal significance associated with the hemocytes of the eastern oyster, C’r?r~~~ea virpinica. METHODS AND RESULTS
Cell sources We obtained our hemocytes from oysters collected from Tampa Bay, Florida. The oysters were maintained in a recirculating artificial seawater system. Cells were obtained by needle-syringe aspiration from the pericardial cavity or an adductor muscle sinus. Cells from several animals were pooled as necessay to obtair! req~lred hemocyte concentrations. Oxvgen uptake by oyster hemocytes Oxygen uptake by piruled hemocyte preparations was recorded using a Clark-type oxygen electrode. Fig. 1 shows a digitized trace taken from a stirred sample of cells in artificial seawater at 27°C. An estimate of O2 uptake for that sample was 13 nmoles/lO min. The uptake of these small amounts of oxygen is reproducible and gives evidence of partial inhibition by cyanide and azide. Hydrogenperoxide productiorl WChave adapted the homovanillic acid method of Nakamura et al. ( 1985) for the measurement of hydrogen peroxide. These workers validated the use
121
OXIDANT PKDDVLTION BY HEMOWTES
of this sensitive fluorescence assay with the scallop Putinopecfen. Fig. 2 shows the results obtained from several combined experiments. Hemocytes (O.l1.0~ 1Q6 cells) were added to reactant mixtures (plus additives as noted). After I h at room temperature (23°C)) the reactions were terminated and the fluorescence of oxidized homovani!!ic acid was measured. Typically each experiment included a time-zero control, 2-3 incremental cell concentrations, and H,O, standards. An endogenous production of H,O, was appzent and the Kmskal-Wallis test (a nonparametric rank test) indicated a significant effect of additives on oxidant production. With the Wilcoxon signed ranks test, significantiy higher concentrations of oxidant were found upon ariditions of Concanavalin A (Con A) and Zymosan. The increase seen with
i
PMA
I-
0.800
1.000
n=6
10 9
a 7 5 5 4 3 2
1 ~ 00000
0.200
0.400
0.600
~e,,s per assay x 106 Fig. 3. H,O, production by ~)ysterhemocyw ! nnnomoles I-LO, vs. cell number-
Con A added).
128
F.E.FRIEDLAND MR. ALVAREZ
Phorbol Myristate Acetate (PMA) was insignificant however. Production of oxidant was found to be proportional to cell concentration (Fig. 3) and ancillary experiments indicated that catalase and oxygen deprivation reduce amounts of oxidant measured. Localization ofperoxidase Using a standard diaminobenzidine method, we found cytoplasmic deposits susesting the presence of peroxidase activity. These deposits, absent in controls, are spheroidal, resembling in shape the granules seen in certain hemocytes. CONCLUSIONS
We have shown that hemocytes of Cmsrorrreu virnlnrcu consume small amounts of oxygen that conceivably could be part of a hydroperoxide metabolism. We detected the endogenous production of an oxidant, presumably H,O,, and showed that more of it appeared wher cells were stimulated. Whether this hydrogen peroxide is associated with cytotoxic effects, an innoo uous consequence of altered cell membrane activity and/or leakage, or simply a normal pattern of oxygen metabolism is not known. Because of the avid
phagocytic activities of many of these cells, the presence of a peroxidase possibly able to potentiate the effects of H202 is of additional interest. We feel that our research strongly suggests the involvement of a hydroperoxide metabolism in biocidal activities of oyster hemocytes. ACKNOWLEDGEMENTS ‘*ve thank Ms. Jacqueline S. Johnson for her expert technical assistance with this research and are indebted to Ms. Carmen S. Patron for her work with hemocyte oxygen uptake. This article was developed under the auspices of
the Florida Sea Grant College Program with support from the National Oceanic and A’mospheric Administration, Oftice of Sea Grant, U.S. Departmeni of Commerce, Grant No. NA86AA-D-SG068. The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon.
REFERENCES
Anderson,R., Holmes,B. and Good, R.. 1973. Comparativebiochemistry of phagocytizing insect hemocytes. Camp.B&hem. Physiol.,46B:595-602. Cheng.T., 1976. Aspectsof substrueutdizationand energyrequirementdunna molluscan phagocytosis. J. Invert.Pathol.,27: 263-268.
OXIDANT
,‘RODUCTlON
BY HEMOWTES
129
Dikkeboom R.. Tijnagel, J., Molder. E. and Van der Knaap. W.. 1987.Hemocees of the pond snail Lymnceo rragnaY generatereactive form5 of oxygen.J. Invert. Pathol., 49: 321-331. Nakamura, M., Mori, K., Inooka, S. knd Nomura, T., 1985. Is vitrc protiction of hydrogen peroxide by the amoebocytesoftbc scallop,Parmoprcren~essoensis (Jay). Cev. Comp. Immunol., 9: 407-417.