Developmental and Comparative Immunology,Vol. 16, pp. 111-122, 1992 Printed in the USA. All rights reserved.
0145-305X/92 $500 + .00 Copyright © 1992 Pergamon Press Ltd.
GENERATION OF REACTIVE OXYGEN METABOLITES BY THE HAEMOCYTES OF THE MUSSEL Mytilus edulis Richard K. Pipe Natural Environment Research Council, Plymouth Marine Laboratory, Citadel Hill, Plymouth PL1 2PB, UK
(Submitted September 1990; Accepted April 1991)
[]Abstract--Generation of superoxide anion by stimulated haemocytes ofMytilus edulis was demonstrated using dihydrorhodamine 123 and quantified using reduction of nitroblue tetrazolium (NBT). In the presence of zymosan or phorbol myristate acetate, there was an increased reduction of NBT to formazan. The addition of superoxide dismutase (SOD) and iodoacetamide to the incubation medium resuited in a significant reduction in deposition of reduced formazan. Incubation of haemocytes with the SOD inhibitor diethyldithiocarbamate (DDC) gave rise to a small but significant increase in NBT reduction. The production of hydrogen peroxide by haemocytes was quantified using horseradish peroxidase-dependent oxidation of phenol red. The presence of SOD in the incubation medium together with zymosan resulted in a significant increase in HzO2 production. Haemocytes incubated with DDC prior to the assay or with sodium nitroprusside during the assay showed a decrease in HzO2 production with increasing concentration of the inhibitor. DKeywords--Bivalve mollusc; Mytilus edulis; Respiratory burst; Haemocytes; Superoxide anion; Hydrogen peroxide; Nitroblue tetrazolium; Phenol red. Introduction
In mammals, the blood p h a g o c y t e s , which include neutrophils, eosinophils, basophils, and monocytes, have a general role in host defense, involving upAddress correspondence to R. K. Pipe, Plymouth Marine Laboratory, Citadel Hill, Plymouth PLI 2PB, UK.
take and digestion of foreign matter and tissue debris, with each cell type performing a different and highly specialized function. All the blood phagocytes contain granules rich in lysosomal enzymes (1-3), which degranulate during phagocytosis to bring about the killing and degradation of endocytosed pathogens. In addition to degranulation and release of hydrolytic enzymes, these cells are capable of an "oxidative or respiratory burst." This comprises a metabolic pathway, generally dormant in resting cells, in which oxygen is partially reduced to a number of highly reactive, microbicidal metabolites including superoxide anion radical (O2-), hydrogen peroxide (H202), and h y d r o x y l radical ( O H ) (4-6). The biochemical basis for the respiratory burst is the activation of a membrane-bound NAD(P)H oxidase in the plasma membrane that catalyzes the univalent reduction of molecular oxygen, forming superoxide anion, which can then be dismutated spontaneously or via catalysis by superoxide dismutase to hydrogen peroxide. Excess hydrogen peroxide may be broken down to H20 and O2 by catalase or alternatively converted to hypochlorous acid (HOC1) via the m y e l o p e r o x i d a s e - - H 2 0 2 - C l - system (7), which also plays an important role in the antimicrobial activity of the phagocytic cells (8,9). Activation of the respiratory burst can be initiated both by physiological processes including phagocytosis of particulates such as bacteria and zymosan or by soluble agents includ-
111
112
ing the protein kinase C activator, phorbol myristate acetate (PMA) (10). In invertebrates, there have been relatively few studies concerning the release of reactive oxygen metabolites from haemocytes, and in molluscs some conflicting results have been reported. Cheng (11) concluded that haemocytes of the clam Mercenaria mercenaria did not produce reactive oxygen metabolites and relied upon release of lysosomal enzymes for microbicidal activity; however, other reports for gastropod and bivalve species have demonstrated release of various components of the respiratory burst (12-15). Previous studies have demonstrated the presence of hydrolytic enzymes in the granular haemocytes of Mytilus edulis (16,17). The purpose of the present study was to investigate whether Mytilus relied exclusively upon release of lysosomal enzymes for antimicrobial activity or if the haemocytes were also able to produce a respiratory burst to aid in destroying potential pathogens. Release of O2- using reduction of nitroblue tetrazolium (18) and of H202 using horseradish peroxidase-dependent oxidation of phenol red (19,20) were investigated.
Materials and Methods Animals Mussels, M. edulis (50- to 60-mm shell length), were collected from an open coastal environment at Whitsand Bay in Cornwall, UK. On transfer to the laboratory, the animals were kept in a recirculating seawater system at seasonal ambient temperature and fed daily on a mixed algal diet. Haemolymph (1 mL) was extracted from the posterior adductor muscle into a 2.5- or 5-mL syringe containing 0.5 mL 0.01 M PBS, 900 mOsm, pH 7.4. EDTA (0.1%) was added to the PBS for experiments on release of
R.K. Pipe
O2-, where haemocytes were kept in suspension, but not for HzO2 production, where the cells were used as monolayers.
Assay for Superoxide Production The reduction of nitroblue tetrazolium (NBT) to insoluble blue formazan has been used widely as a probe for superoxide anion generation although it is not entirely specific for 0 2 - (21,22). The reaction can, however, be used to quantify the O2- generated in systems where the production of the O2- has been independently assessed (23). To validate the 0 2 generation by mussel haemocytes, the nonfluorescent probe dihydrorhodamine 123 (DHR) was employed. This probe is an uncharged, nontoxic derivative of rhodamine 123, which is oxidised intracellularly by superoxide anion during the respiratory burst to brightly fluorescent rhodamine 123 (24,25); under physiological conditions, this oxidation is specific for 0 2 - (26). Samples of haemolymph were extracted into either PBS or Bakers formol calcium and DHR (Molecular Probes Inc.) added to give a concentration of 43 IxM in the presence and absence of zymosan (0.025%). Bright green fluorescence (excitation at 485 nm) was observed for live cells in the presence of zymosan, with minimal fluorescence in the absence of zymosan and no fluorescence with the fixed cells. Having established that the haemocytes generate 0 2 when stimulated, this was then quantified using the NBT assay as follows. Haemolymph (0.5 mL) was incubated in an equal volume of medium consisting of PBS, pH 7.4, 900 mOsm containing 1% bovine serum albumin (BSA), 2 mM CaCI 2, and 0.17% NBT. Incubations were carried out at 20°C in a dark, humid environment for between 1 and 4 h in the presence or absence of the respiratory burst stimuli, zymosan and PMA. Following incubation, the haemocytes were
Reactive oxygen metabolites in mussel haemocytes
fixed by the addition of concentrated formalin (4% final concentration) and optical density readings taken on individual haemocytes (3 replicates of 50 readings per treatment) using a Vickers M85 scanning microdensitometer at a wavelength of 580 nm with a spot size of 1 Ixm, a mask area of 314 ~m 2, and an X40 objective. The effects on O2- production of incubating the h a e m o c y t e s with 300 U m L -~ superoxide dismutase (SOD) (Sigma), the SOD inhibitor sodium nitroprusside (I mM) (27), and the respiratory burst inhibitor iodoacetamide (I0 mM) (18) were investigated. The SOD inhibitor d i e t h y l d i t h i o c a r b a m a t e ( D D C )
113
(28,29) at 10 mM concentration was incubated with the haemocytes for 3 h prior to the O2- determination, as DDC itself has a reducing effect. Exposure to DDC was followed by three washes in PBS, controls were preincubated in PBS.
Assay for Hydrogen Peroxide Production
Aliquots of haemolymph (I00 IxL) from individual mussels were pipetted into 96-well microtitre plates and left for 2 h in a dark, humid environment for the haemocytes to adhere. Immediately fol-
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Time of Incubation (hours) Figure 1. Effects of zymosan on the reduction of NBT by haemocytes from M. edulis. Values are means of 100 readings on individual haemocytes - 2 SE. (A), without NBT or zymosan; (B), with NBT without zymosan; (C) with NBT and zymosan (0.025%).
114
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ings were determined in a Multiskan microtitre plate reader at 620 nm. The optical density readings were converted to ~M H202 using a standard curve established from solutions of known H202 molarity. To convert values to IxM H202 per p~g protein, four extra wells of haemocyte monolayers were prepared for each animal and subsequently the mean protein level determined using a bicinchoninic acid (BCA) protein assay (Pierce Chemical Co.). The cells were solubilized using the detergent CHAPS at 1% (Pierce Chemical Co.) or overnight incubation in 1 N NaOH at 37°C and the protein concentration determined using a
lowing the removal of plasma and nonadherent cells, the monolayers were covered with 100 p,L phenol red solution (PRS). The PRS contained PBS pH 7.4, 900 mOsm, 5.5 mM dextrose (BDH Chemicals Ltd.), 0.56 mM phenol red (Sigma), and 8.5 U m L -1 horseradish peroxidase (type II, Sigma). Prior to the assay, z y m o s a n (0.05%), SOD (150 U m L - ~ ) , and sodium nitroprusside could be added to the PRS. The SOD inhibitor DDC was incubated with the cells for 3 h followed by three washes in PBS before the assay for H202. The reaction was stopped by the addition of 30 IxL IN NaOH, and optical density read-
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Time of I n c u b a t i o n (hours) Figure 2. Effects of PMA on the reduction of NBT by haemocytes from M. edulis. Values are means
of 150 readings on individual haemocytes - 1 SE. (A), without PMA; (B), with 0.2 l~g mL -1 PMA; (C), with 5.0 i~g mL -~ PMA.
Reactive oxygen metabolites in mussel haemocytes
standard curve prepared with BSA. Readings were done at 540 nm using the microtitre plate reader.
Results Reduction of NBT Deposits of blue formazan were seen in haemocytes incubated in the presence o f NBT. M e a s u r e m e n t s o v e r time showed an increase in formazan deposition. H a e m o c y t e s incubated without NBT showed no change in optical density readings over a 4-h incubation pe-
115
riod (Fig. I). In the presence of zymosan, there was an increased reduction of NBT to formazan (Fig. 1); similar results were obtained using a soluble stimulus, PMA (Fig. 2). The controls for experiments with zymosan and PMA were similar [Fig. I(B) cf Fig. 2(A)], both showing increasing amounts of reduced NBT with time; maximum values did not reach those obtained with the addition of a stimulus. This spontaneous reduction of NBT in the controls indicates that experimental conditions in themselves elicit a response from the cells. The concentration of PMA resulting in the maximum
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C o n c e n t r a t i o n of PMA (~g ml "1) Figure 3. Effects of different concentrations of PMA on the reduction of NBT by haemocytes from M. edulis incubated for different times. Values are means of 150 readings on individual haemocytes
+- 2 SE. (A), incubated for 30 min; (B), incubated for 90 min; (C), incubated for 180 min; (D), incubated for 240 min.
116
R.K. Pipe
reduction of NBT was between 0.5 and 5.0 I~gmL-~ for all incubations between 30 min and 4 h (Fig. 3). The addition of SOD or iodoacetamide to the incubation medium led to a significant reduction in deposition of reduced formazan for all time intervals investigated (Fig. 4). The SOD inhibitor sodium nitroprusside also, unexpectedly, resulted in a significant reduction in reduced formazan production for incubations in excess of 1 h (Fig. 4). Incubation of the haemocytes with the SOD inhibitor DDC resulted in a small but significant (three-way analysis of variance
12
significant difference p < 0.05) increase in NBT reduction for all incubations times (Fig. 5).
Oxidation of Phenol Red Incubation of haemocytes with phenol red and horseradish peroxidase in the presence of zymosan resulted in an increase in absorbance, when read at 620 nm, for time intervals up to 25 min, after which time values decreased (Fig. 6). Unlike the NBT reduction, haemocytes incubated without zymosan did not show
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Time of Incubation (hours) Figure 4. Effects on reduction of NBT by haemocytes from M. edufis by incubation with inhibitors and exogenous SOD, Values are means of 150 readings on individual haemocytes +- 2 SE. (A),
iodoacetamide (10 mM); (B), SOD (300 U mL-1); (C), sodium nitroprusside (1 mM); (D), control.
Reactive oxygen metabolites in mussel haemocytes
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0145-305X/92 $500 + .00 Copyright © 1992 Pergamon Press Ltd.
GENERATION OF REACTIVE OXYGEN METABOLITES BY THE HAEMOCYTES OF THE MUSSEL Mytilus edulis Richard K. Pipe Natural Environment Research Council, Plymouth Marine Laboratory, Citadel Hill, Plymouth PL1 2PB, UK
(Submitted September 1990; Accepted April 1991)
[]Abstract--Generation of superoxide anion by stimulated haemocytes ofMytilus edulis was demonstrated using dihydrorhodamine 123 and quantified using reduction of nitroblue tetrazolium (NBT). In the presence of zymosan or phorbol myristate acetate, there was an increased reduction of NBT to formazan. The addition of superoxide dismutase (SOD) and iodoacetamide to the incubation medium resuited in a significant reduction in deposition of reduced formazan. Incubation of haemocytes with the SOD inhibitor diethyldithiocarbamate (DDC) gave rise to a small but significant increase in NBT reduction. The production of hydrogen peroxide by haemocytes was quantified using horseradish peroxidase-dependent oxidation of phenol red. The presence of SOD in the incubation medium together with zymosan resulted in a significant increase in HzO2 production. Haemocytes incubated with DDC prior to the assay or with sodium nitroprusside during the assay showed a decrease in HzO2 production with increasing concentration of the inhibitor. DKeywords--Bivalve mollusc; Mytilus edulis; Respiratory burst; Haemocytes; Superoxide anion; Hydrogen peroxide; Nitroblue tetrazolium; Phenol red. Introduction
In mammals, the blood p h a g o c y t e s , which include neutrophils, eosinophils, basophils, and monocytes, have a general role in host defense, involving upAddress correspondence to R. K. Pipe, Plymouth Marine Laboratory, Citadel Hill, Plymouth PLI 2PB, UK.
take and digestion of foreign matter and tissue debris, with each cell type performing a different and highly specialized function. All the blood phagocytes contain granules rich in lysosomal enzymes (1-3), which degranulate during phagocytosis to bring about the killing and degradation of endocytosed pathogens. In addition to degranulation and release of hydrolytic enzymes, these cells are capable of an "oxidative or respiratory burst." This comprises a metabolic pathway, generally dormant in resting cells, in which oxygen is partially reduced to a number of highly reactive, microbicidal metabolites including superoxide anion radical (O2-), hydrogen peroxide (H202), and h y d r o x y l radical ( O H ) (4-6). The biochemical basis for the respiratory burst is the activation of a membrane-bound NAD(P)H oxidase in the plasma membrane that catalyzes the univalent reduction of molecular oxygen, forming superoxide anion, which can then be dismutated spontaneously or via catalysis by superoxide dismutase to hydrogen peroxide. Excess hydrogen peroxide may be broken down to H20 and O2 by catalase or alternatively converted to hypochlorous acid (HOC1) via the m y e l o p e r o x i d a s e - - H 2 0 2 - C l - system (7), which also plays an important role in the antimicrobial activity of the phagocytic cells (8,9). Activation of the respiratory burst can be initiated both by physiological processes including phagocytosis of particulates such as bacteria and zymosan or by soluble agents includ-
111
112
ing the protein kinase C activator, phorbol myristate acetate (PMA) (10). In invertebrates, there have been relatively few studies concerning the release of reactive oxygen metabolites from haemocytes, and in molluscs some conflicting results have been reported. Cheng (11) concluded that haemocytes of the clam Mercenaria mercenaria did not produce reactive oxygen metabolites and relied upon release of lysosomal enzymes for microbicidal activity; however, other reports for gastropod and bivalve species have demonstrated release of various components of the respiratory burst (12-15). Previous studies have demonstrated the presence of hydrolytic enzymes in the granular haemocytes of Mytilus edulis (16,17). The purpose of the present study was to investigate whether Mytilus relied exclusively upon release of lysosomal enzymes for antimicrobial activity or if the haemocytes were also able to produce a respiratory burst to aid in destroying potential pathogens. Release of O2- using reduction of nitroblue tetrazolium (18) and of H202 using horseradish peroxidase-dependent oxidation of phenol red (19,20) were investigated.
Materials and Methods Animals Mussels, M. edulis (50- to 60-mm shell length), were collected from an open coastal environment at Whitsand Bay in Cornwall, UK. On transfer to the laboratory, the animals were kept in a recirculating seawater system at seasonal ambient temperature and fed daily on a mixed algal diet. Haemolymph (1 mL) was extracted from the posterior adductor muscle into a 2.5- or 5-mL syringe containing 0.5 mL 0.01 M PBS, 900 mOsm, pH 7.4. EDTA (0.1%) was added to the PBS for experiments on release of
R.K. Pipe
O2-, where haemocytes were kept in suspension, but not for HzO2 production, where the cells were used as monolayers.
Assay for Superoxide Production The reduction of nitroblue tetrazolium (NBT) to insoluble blue formazan has been used widely as a probe for superoxide anion generation although it is not entirely specific for 0 2 - (21,22). The reaction can, however, be used to quantify the O2- generated in systems where the production of the O2- has been independently assessed (23). To validate the 0 2 generation by mussel haemocytes, the nonfluorescent probe dihydrorhodamine 123 (DHR) was employed. This probe is an uncharged, nontoxic derivative of rhodamine 123, which is oxidised intracellularly by superoxide anion during the respiratory burst to brightly fluorescent rhodamine 123 (24,25); under physiological conditions, this oxidation is specific for 0 2 - (26). Samples of haemolymph were extracted into either PBS or Bakers formol calcium and DHR (Molecular Probes Inc.) added to give a concentration of 43 IxM in the presence and absence of zymosan (0.025%). Bright green fluorescence (excitation at 485 nm) was observed for live cells in the presence of zymosan, with minimal fluorescence in the absence of zymosan and no fluorescence with the fixed cells. Having established that the haemocytes generate 0 2 when stimulated, this was then quantified using the NBT assay as follows. Haemolymph (0.5 mL) was incubated in an equal volume of medium consisting of PBS, pH 7.4, 900 mOsm containing 1% bovine serum albumin (BSA), 2 mM CaCI 2, and 0.17% NBT. Incubations were carried out at 20°C in a dark, humid environment for between 1 and 4 h in the presence or absence of the respiratory burst stimuli, zymosan and PMA. Following incubation, the haemocytes were
Reactive oxygen metabolites in mussel haemocytes
fixed by the addition of concentrated formalin (4% final concentration) and optical density readings taken on individual haemocytes (3 replicates of 50 readings per treatment) using a Vickers M85 scanning microdensitometer at a wavelength of 580 nm with a spot size of 1 Ixm, a mask area of 314 ~m 2, and an X40 objective. The effects on O2- production of incubating the h a e m o c y t e s with 300 U m L -~ superoxide dismutase (SOD) (Sigma), the SOD inhibitor sodium nitroprusside (I mM) (27), and the respiratory burst inhibitor iodoacetamide (I0 mM) (18) were investigated. The SOD inhibitor d i e t h y l d i t h i o c a r b a m a t e ( D D C )
113
(28,29) at 10 mM concentration was incubated with the haemocytes for 3 h prior to the O2- determination, as DDC itself has a reducing effect. Exposure to DDC was followed by three washes in PBS, controls were preincubated in PBS.
Assay for Hydrogen Peroxide Production
Aliquots of haemolymph (I00 IxL) from individual mussels were pipetted into 96-well microtitre plates and left for 2 h in a dark, humid environment for the haemocytes to adhere. Immediately fol-
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Time of Incubation (hours) Figure 1. Effects of zymosan on the reduction of NBT by haemocytes from M. edulis. Values are means of 100 readings on individual haemocytes - 2 SE. (A), without NBT or zymosan; (B), with NBT without zymosan; (C) with NBT and zymosan (0.025%).
114
R.K. Pipe
ings were determined in a Multiskan microtitre plate reader at 620 nm. The optical density readings were converted to ~M H202 using a standard curve established from solutions of known H202 molarity. To convert values to IxM H202 per p~g protein, four extra wells of haemocyte monolayers were prepared for each animal and subsequently the mean protein level determined using a bicinchoninic acid (BCA) protein assay (Pierce Chemical Co.). The cells were solubilized using the detergent CHAPS at 1% (Pierce Chemical Co.) or overnight incubation in 1 N NaOH at 37°C and the protein concentration determined using a
lowing the removal of plasma and nonadherent cells, the monolayers were covered with 100 p,L phenol red solution (PRS). The PRS contained PBS pH 7.4, 900 mOsm, 5.5 mM dextrose (BDH Chemicals Ltd.), 0.56 mM phenol red (Sigma), and 8.5 U m L -1 horseradish peroxidase (type II, Sigma). Prior to the assay, z y m o s a n (0.05%), SOD (150 U m L - ~ ) , and sodium nitroprusside could be added to the PRS. The SOD inhibitor DDC was incubated with the cells for 3 h followed by three washes in PBS before the assay for H202. The reaction was stopped by the addition of 30 IxL IN NaOH, and optical density read-
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Time of I n c u b a t i o n (hours) Figure 2. Effects of PMA on the reduction of NBT by haemocytes from M. edulis. Values are means
of 150 readings on individual haemocytes - 1 SE. (A), without PMA; (B), with 0.2 l~g mL -1 PMA; (C), with 5.0 i~g mL -~ PMA.
Reactive oxygen metabolites in mussel haemocytes
standard curve prepared with BSA. Readings were done at 540 nm using the microtitre plate reader.
Results Reduction of NBT Deposits of blue formazan were seen in haemocytes incubated in the presence o f NBT. M e a s u r e m e n t s o v e r time showed an increase in formazan deposition. H a e m o c y t e s incubated without NBT showed no change in optical density readings over a 4-h incubation pe-
115
riod (Fig. I). In the presence of zymosan, there was an increased reduction of NBT to formazan (Fig. 1); similar results were obtained using a soluble stimulus, PMA (Fig. 2). The controls for experiments with zymosan and PMA were similar [Fig. I(B) cf Fig. 2(A)], both showing increasing amounts of reduced NBT with time; maximum values did not reach those obtained with the addition of a stimulus. This spontaneous reduction of NBT in the controls indicates that experimental conditions in themselves elicit a response from the cells. The concentration of PMA resulting in the maximum
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C o n c e n t r a t i o n of PMA (~g ml "1) Figure 3. Effects of different concentrations of PMA on the reduction of NBT by haemocytes from M. edulis incubated for different times. Values are means of 150 readings on individual haemocytes
+- 2 SE. (A), incubated for 30 min; (B), incubated for 90 min; (C), incubated for 180 min; (D), incubated for 240 min.
116
R.K. Pipe
reduction of NBT was between 0.5 and 5.0 I~gmL-~ for all incubations between 30 min and 4 h (Fig. 3). The addition of SOD or iodoacetamide to the incubation medium led to a significant reduction in deposition of reduced formazan for all time intervals investigated (Fig. 4). The SOD inhibitor sodium nitroprusside also, unexpectedly, resulted in a significant reduction in reduced formazan production for incubations in excess of 1 h (Fig. 4). Incubation of the haemocytes with the SOD inhibitor DDC resulted in a small but significant (three-way analysis of variance
12
significant difference p < 0.05) increase in NBT reduction for all incubations times (Fig. 5).
Oxidation of Phenol Red Incubation of haemocytes with phenol red and horseradish peroxidase in the presence of zymosan resulted in an increase in absorbance, when read at 620 nm, for time intervals up to 25 min, after which time values decreased (Fig. 6). Unlike the NBT reduction, haemocytes incubated without zymosan did not show
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Time of Incubation (hours) Figure 4. Effects on reduction of NBT by haemocytes from M. edufis by incubation with inhibitors and exogenous SOD, Values are means of 150 readings on individual haemocytes +- 2 SE. (A),
iodoacetamide (10 mM); (B), SOD (300 U mL-1); (C), sodium nitroprusside (1 mM); (D), control.
Reactive oxygen metabolites in mussel haemocytes
117
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