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Effect of Tetramicra brevifilum (Microspora) infection on respiratory-burst responses of turbot (Scophthalmus maximus L.) phagocytes
Fish & Shellfish Immunology (2001) 11, 639–652 doi:10.1006/fsim.2001.0340 Available online at http://www.idealibrary.com on
Effect of Tetramicra brevifilum (Microspora) infection on respiratory-burst responses of turbot (Scophthalmus maximus L.) phagocytes J. LEIRO1*, R. IGLESIAS2, A. PARAMAu 2, M. L. SANMARTIuN2
AND
F. M. UBEIRA1
Departamento de Microbiología y Parasitología and 2Instituto de Investigación y Análisis Alimentarios, Laboratorio de Parasitología, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain 1
(Received 1 November 2000, accepted after revision 23 January 2001, published electronically 3 August 2001) In vitro assays were performed to investigate microsporidian-induced intracellular and extracellular production of reactive oxygen species (ROS) by peritoneal-exudate adherent (PEA) cells from turbot. ROS production was quantified using the fluorescent reagents OxyBURST Green H2HFF BSA (extracellular) and OxyBURST Green H2DCFDA succinimidyl ester (intracellular). Five days before assay, the cells had been elicited in vivo by intraperitoneal injection of sodium thioglycollate or spores of Tetramicra brevifilum. Elicitation with spores led to a marked increase in the proportion of neutrophils among PEA cells. PEA cells from normal turbot showed considerable extracellular and intracellular ROS production in response to microsporidian spores. By contrast, PEA cells from microsporidian-infected turbot showed considerably reduced extracellular and intracellular ROS production in response to microsporidian spores. Extracellular ROS production was a#ected by the addition of infected turbot serum to the assay medium, regardless of whether the PEA cells had been obtained from normal or infected fish. The presence of microsporidian-infected turbot serum significantly reduced intracellular ROS production by PEA cells elicited with microsporidian spores. These results suggest that (a) microsporidian spores partially suppress the repiratory-burst response of turbot phagocytes; and (b) infected turbot serum contains substances capable of modulating the respiratory-burst response of turbot phagocytes to microsporidian spores. 2001 Academic Press Key words:
turbot, microsporidians, neutrophils.
respiratory-burst,
macrophages,
I. Introduction Of the parasites capable of infecting farmed turbot, microsporidians are probably those that provoke the greatest losses (Person-Le Ruyet, 1990; Estévez et al., 1992; Figueras et al., 1992; Lom & Dyková, 1992). Microsporidians are primitive eukaryotic intracellular parasites (Vossbrinck et al., 1987). In fish, very little is known about their biology or their immunobiology (Lom & Dyková, 1992). Currently, the only species of micro*Corresponding author. E-mail: [email protected] 1050–4648/01/070639+14 $35.00/0
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sporidian recognised as a pathogen of the turbot is Tetramicra brevifilum (Matthews & Matthews, 1980). The biology of this species within turbot has been characterised in detail (Matthews & Matthews, 1980), but the rest of its life-cycle is not known. In addition, very limited information is available on the immunobiology of turbot infected by this parasite, although there have been studies on humoral immune responses in natural infections (Leiro et al., 1993, 1996a) and of in vitro non-specific responses of turbot immune cells to microsporidian spores (Leiro et al., 1996b, 1997, 2000). During microsporidian infection in fishes, phagocytosis constitutes the final stage of defence and plays an important role in destruction of the spores (Dyková & Lom, 1980; Pulsford & Matthews, 1991; Lom & Dyková, 1992). Generally, after internalisation of the parasite by a phagocyte, it is destroyed by toxic oxygen metabolites produced during the respiratory burst (Babior, 1978; Mauël, 1982; Secombes & Fletcher, 1992). Like other intracellular protozoa, however, microsporidians may replicate within the parasitophorous vacuoles of phagocytes (Weidner, 1975; Didier, 1995). In mammals, some microsporidians are able to survive in the intracellular location, probably because of their capacity to inhibit the respiratory burst, or to avoid in other ways the e#ects of phagosome–lysosome fusion (Mauël, 1982; Schmidt & Shadduck, 1984; Didier et al., 1994; Didier, 1995). In the current study, we used a sensitive fluorescent assay to monitor intracellular and extracellular reactive oxygen species (ROS) production by adherent cells from inflamed-peritoneal exudates of normal and naturally infected turbot, and evaluated the possible modulatory e#ects of components present in the serum of normal and naturally infected turbot on the respiratory burst of phagocytes. II. Materials and Methods FISH AND SERA
Uninfected juvenile turbot Scophthalmus maximus L. (50–100 g) were obtained from a local farm (Insuin˜ a, El Grove, Pontevedra, north-western Spain). Naturally infected turbot (50–100 g) were obtained from a fish farm in the north of Galicia (north-western Spain). Prior to experiments, the fish were acclimatised for at least 15 days in 10 l tanks with a constant flow of water (181 C, pH 6·50·5) and aeration. The fish received a standard semi-dried pelleted food daily. Blood samples were collected by caudal vein puncture. The serum was separated by centrifugation at 2000g for 10 min and stored at 30 C until use.
PARASITES
Viable microsporidian spores (T. brevifilum, Matthews & Matthews, 1980) were isolated from naturally infected turbot, as described previously (Estévez et al., 1992), and stored at 4 C in sterile sea water containing 0·1% sodium
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azide. Spore viability was determined by the method of Modha et al. (1997), which is based on the incorporation of fluorescent compounds into the spore wall (Leiro et al., 2000). ELICITATION OF INFLAMMATORY CELLS
Inflammatory cells were elicited by i.p. injection of 1 ml of 3% Brewer thioglycollate medium, or 109 T. brevifilum spores suspended in PBS, into anaesthetised turbot (0·03% 2-phenoxy ethanol, Sigma), and harvested 5–6 days later. COLLECTION OF PERITONEAL LEUCOCYTES
The fish were anaesthetised as indicated above, and the anaesthesia was prolonged until death. The abdominal side of the fish was wetted with 70% ethanol, and the fish were injected through the peritoneal wall at the midline using a 10 ml syringe attached to a 22 G needle, with 5–10 ml (depending on the size of the fish) of phenol-red-free Hanks’ balanced salts solution (HBSS) containing Ca2+ and Mg2+ . Using the same syringe and needle, peritoneal fluid was then slowly withdrawn and pooled in 50 ml polypropylene centrifuge tubes on ice. PREPARATION OF PERITONEAL EXUDATE ADHERENT CELLS
Cell concentration was counted using a haemocytometer and adjusted in HBSS to 106 cells ml 1 by centrifugation at 500g for 15 min at 4 C. Aliquots of the cell suspension (100 l) were then added to the wells of 96 well microculture plates (Corning, U.S.A.) and left for 60 min at 23 C to allow adhesion. Non-adherent cells were then removed by two washes with 200 l well 1 of HBSS. The number of viable adherent cells was determined using the trypan blue exclusion test. Trypan blue (0·4% in PBS) was added to wells and incubated for 3 min at room temperature, after which the number of unstained (viable) and stained (non-viable) cells was counted. QUANTIFICATION OF EXTRACELLULAR RELEASE OF ROS
The fluorogenic reagent OxyBURST Green H2HFF BSA (Molecular Probes, The Netherlands) was used for the detection of extracellular ROS release by peritoneal-exudate adherent (PEA) cells (Ryan et al., 1990). This reagent consists of bovine serum albumin coupled to dihydro-2 ,4,5,6,7,7 hexafluorofluorescein (H2HFF), which becomes fluorescent on oxidation. BSA is a large protein, so the reagent does not cross the cell membrane, ensuring detection of extracellular oxidants only. For the assay, 100 l of PEA cells (106 cells ml 1) were incubated with microsporidian spores (cell:spore ratio 1:85, Leiro et al., 1995) for 1 h at 23 C, washed twice with HBSS and incubated in HBSS containing 10 g ml 1 of OxyBURST H2HFF Green BSA in a 96 well microculture plate. The release of oxidative products was quantified in a microplate fluorescence reader (Bio-Tek Instruments, U.K.; excitation 488 nm, detection 530 nm) as the increase in fluorescence over a 55 min period. QUANTIFICATION OF INTRACELLULAR RELEASE OF ROS
For the detection of intracellular ROS production by PEA cells, we used the amine-reactive fluorogenic reagent OxyBURST Green H2DCFDA
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succinimidyl ester (Molecular Probes, The Netherlands). Following conjugation to amines, the acetates of this reagent are removed by treatment with hydroxylamine to yield the H2DCF conjugate. As the H2DCF moiety is non-fluorescent until oxidised to dichlorofluorescein when the conjugated particles are internalised within the phagovacuole, this reagent is particularly suitable for detecting the intracellular oxidative burst in phagocytic cells (Cao et al., 1993). For microsporidian spore labelling, 100 l of the OxyBURST reagent (10 mg ml 1 in dimethylsulfoxide, DMSO) was added to a suspension of 10 mg of spores in 1 ml of 0·05 M borate bu#er pH 8·3, and incubated for 1 h in the dark at room temperature with continuous gentle shaking. To remove the acetates, 0·1 ml of 1·5 M hydroxylamine pH 8·5 was then added to the reaction mixture, which was incubated for 1 h at room temperature with stirring, then overnight at 4 C without stirring. Finally, the conjugate was separated from the unreacted reagent by repeated centrifugation in PBS at 10 000g for 3 min, until fluorescence in the supernatant dropped to zero. The spores were stored in the dark at 4 C in PBS containing 0·01% sodium azide. For the assay, 1 l aliquots of the spore suspension (previously centrifuged at 10 000g for 3 min to remove the sodium azide) were added to the wells of microculture plates containing PEA cells. Fluorescence emission was then determined in a microplate fluorescence reader (Bio-Tek Instruments; excitation 488 nm, detection 530 nm) over a 55 min period. CYTOCHEMISTRY
Peroxidase staining PEA cells on microscope slides were fixed with 10% formol-ethanol (10 ml of 37% formaldehyde and 90 ml of absolute ethanol) for 60 s at room temperature (Kaplow, 1965), then gently washed with distilled water and incubated for 15 min in TBS bu#er (50 mM Tris, 0·15 M NaCl, pH 7·4) containing 0·003% H2O2, 0·06% diaminobenzidine tetrahydrochloride and 0·03% NiCl2. The slides were then washed with TBS and contrasted with Giemsa stain for 10 min. After washing with distilled water, the slides were air-dried and permanent mounts were prepared with Eukitt. Controls were performed by omitting hydrogen peroxide from the incubation mixture. Stained cells (dark brown cytoplasm) were counted as peroxidase-positive. Non-specific esterase staining Macrophages were identified among PEA cells by determination of acid -naphthyl acetate esterase activity (ANAE staining; Ennist & Jones, 1983), as described previously (Leiro et al., 2000). DATA PRESENTATION AND STATISTICAL ANALYSIS
Intracellular formation/extracellular release of ROS were expressed in terms of variation of fluorescence (in arbitrary units) per unit of time (min). Results shown in the figures are expressed as means S.E. of three replicate
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Percentage of peritoneal– exudate adherent cells
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Fig. 1. Percentages of PEA cells showing -naphthyl acetate esterase (ANAE) activity ( ) or peroxidase activity ( ). Results are shown for cells elicited in vivo with T. brevifilum spores (A) or with sodium thioglycollate (B). There was a statistically significant di#erence between A and B (P<0·01) in the proportion of cells with peroxidase activity, but not in the proportion of cells with ANAE activity.
assays, each using the pooled adherent cells from five turbot. Significant di#erences between two means (P<0·05 or P<0·01) were determined by unpaired Student’s two-tailed t-test. The Welch t-test was used when the populations showed significant di#erence in S.D. III. Results PEROXIDASE AND NON-SPECIFIC ESTERASE ACTIVITIES OF PERITONEAL-EXUDATE ADHERENT CELLS
Peroxidase and non-specific esterase staining was used to identify cell types among the PEA cells elicited by i.p. injection, 5 days previously, of sodium thioglycollate or T. brevifilum spores. Cells showing peroxidase activity were classed as neutrophils, and cells showing nonspecific esterase activity as macrophages. Fig. 1 shows the percentages of PEA cells in each category. Nearly all cells were either macrophages or neutrophils. The proportion of neutrophils was much higher in fish injected with microsporidian spores than in fish injected with thioglycollate. EXTRACELLULAR RELEASE OF ROS BY PEA CELLS
Fig. 2 shows the results of assays of extracellular release of reactive oxygen species by PEA cells incubated with (a) microsporidian spores alone, (b) microsporidian spores plus normal turbot serum, (c) microsporidian spores plus infected turbot serum, or (d) microsporidian spores plus the ROSscavenger L(+) ascorbic acid. The PEA cells were from fish that had been injected 5 days previously with sodium thioglycollate (Fig. 2a) or T. brevifilum spores (Fig. 2b). The trypan blue exclusion test indicated that viabilities were 903% for PEA cells incubated with microsporidian spores alone, 953% for PEA cells incubated with spores plus normal turbot serum, and 925% for
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Fig. 2. Extracellular ROS production (i.e. fluorescence emission by oxidised OxyBURST Green H2HFF BSA) by PEA cells obtained from normal turbot (a) and (b) and microsporidian-infected turbot (c) and (d) after in vivo elicitation with sodium thioglycollate (a) and (c) or T. brevifilum spores (b) and (d). The PEA cells were incubated at 23 C in Hanks’ balanced salt solution with microsporidian spores ( ) and, in some experiments, 10% normal turbot serum (NTS) ( ) or microsporidianinfected turbot serum (ITS) ( ). For negative controls, 100 M L(+) ascorbic acid was included in the incubation medium (). Values shown are the means of increase of fluorescence (in arbitrary units) min1 S.E. of three replicate assays. Asterisks indicate a statistically significant di#erence (P<0·05) with respect to the corresponding value obtained after stimulation with T. brevifilum spores.
PEA cells incubated with spores plus microsporidian-infected turbot serum: in other words, none of the assay components were significantly cytotoxic. Net ROS release was markedly reduced by the inclusion of L(+) ascorbic acid in the reaction mixture, regardless of whether elicitation had been with thioglycollate or microsporidian spores (Fig. 2a and b). In assays with PEA cells elicited in vivo with thioglycollate, the addition of infected turbot serum likewise markedly reduced net ROS release (Fig. 2a). In assays of PEA cells elicited in vivo with microsporidian spores, the addition of turbot serum had no e#ect on ROS release (Fig. 2b). Fig. 2 (c and d) shows the results of assays of extracellular release of ROS by PEA cells from naturally T. brevifilum infected turbot where the PEA cells had been elicited in vivo 5 days previously with thioglycollate (Fig. 2c) or T. brevifilum spores (Fig. 2d). The fish used in this experiment were naturally infected, with large numbers of xenomas observable in the musculature, and a
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Fig. 3. Intracellular ROS production (i.e. fluorescence emission by oxidised OxyBURST Green H2DCFDA succinimidyl ester) by PEA cells obtained from normal turbot (a) and (b) or microsporidian-infected turbot (c) and (d) after in vivo elicitation with sodium thioglycollate (a) and (c) or T. brevifilum spores (b) and (d). The PEA cells were incubated with spores of T. brevilfilum ( ) and, in some experiments, 10% normal turbot serum (NTS) ( ) or microsporidian-infected turbot serum (ITS) ( ). For negative controls, 150 mM sodium azide was included in the incubation medium ( ), and without PEA cells but with spores H2DCF (). All values shown are the mean of fluorescence increase (in arbitrary units) per min S.E. after subtraction of those obtained in identical assays but without spores in the incubation medium. Asterisks indicate significant di#erences (*P<0·05; **P<0·01) with respect to the value obtained after stimulation with microsporidian spores.
mean infection intensity of 16106 spores g1 of muscle (range 2105– 4107). Extracellular ROS production in response to spores alone by PEA cells from infected fish following elicitation with thioglycollate was very low (Fig. 2c), and the response following elicitation with microsporidian spores was only slightly higher (Fig. 2d). In all cases, ROS production was reduced by the addition of infected turbot serum to the assay mixture (Fig. 2c and d). INTRACELLULAR RELEASE OF ROS BY PEA CELLS
Fig. 3 shows the results of assays of intracellular (i.e. withinphagolysosome) release of ROS by PEA cells from normal turbot (Fig. 3a and b) and T. brevifilum infected turbot (Fig. 3c and d) 5 days after elicitation with thioglycollate (Fig. 3a and c) or T. brevifilum spores (Fig. 3b or d). As in the previous experiments, PEA cells were incubated with (a) T. brevifilum spores
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alone, (b) microsporidian spores plus normal turbot serum or (d) microsporidian spores plus 1% sodium azide (negative control, no ROS production). Net ROS release by PEA cells from normal turbot was much higher after elicitation with microsporidian spores than after elicitation with thioglycollate (Fig. 3b and a, respectively). In assays with PEA cells obtained from normal turbot after elicitation with microsporidian spores, the presence of infected turbot serum led to reduced ROS release (Fig. 3b) and the presence of infected turbot serum had a significant e#ect on ROS release by PEA cells from infected turbot (Fig. 3d). IV. Discussion In fish, both macrophages and neutrophils can generate ROS (Secombes, 1996; Dalmo et al., 1997); indeed, in some species, for example Atlantic salmon, the production of O 2 following stimulation with phorbol esters is greater from neutrophils than macrophages (Lamas & Ellis, 1994). It has also been demonstrated that not only macrophages, but also neutrophils show phagocytic activity in fishes (Ainsworth & Dexiang, 1990), including the turbot (Leior et al., 2000). Furthermore, the number of neutrophils is greatly increased during inflammation induced by phlogistic agents (e.g. India ink, casein, incomplete Freund’s adjuvant, formol-killed bacteria; MacArthur et al., 1984; Hine, 1992; Afonso et al., 1998a,b), by phorbol esters (Ainsworth et al., 1996) or by microsporidian infections (Amigó et al., 1996; Leiro et al., 1999). The xenoma-production phase of microsporidian infection provokes proliferative inflammation (Lom & Dyková, 1992), characterised by the presence of fibroblasts and phagocytic cells (largely macrophages and neutrophils) (Pulsford & Matthews, 1991; Lom & Dyková, 1992; Leiro et al., 1999). In the present study, the injection of turbot with microsporidian spores greatly increased the production of neutrophils among the PEA cells. It has been demonstrated that some microsporidians can inhibit phagolysosome formation (Weidner, 1975; Schmidt & Shadduck, 1984), and that macrophages phagocytosing microsporidian spores may also secrete prostaglandins which suppress the immune response in fish (Laudan et al., 1987). To date, however, there have been no studies of the possible mechanisms by which microsporidians can escape the phagocyte respiratory burst. In principle, two possible mechanisms might be hypothesised: (i) non-elicitation of the respiratory burst (Mauël, 1997), and (ii) neutralisation of the ROS produced during the respiratory burst. In the present study, we investigated the in vitro production of ROS, in response to microsporidian spores, by PEA cells that had been elicited in vivo with either microsporidian spores or thioglycollate. Both extracellular and intracellular ROS production were investigated, using fluorogenic reagents. For negative controls in assays of extracellular ROS production, we used L(+) ascorbic acid, which is a potent scavenger of reactive oxygen and nitrogen species (Carr & Frei, 1999). For negative controls in assays of intracellular ROS production, we used sodium azide, which is a potent inhibitor of oxidative phosphorylation and catalase activity, and thus inhibits ROS release by quenching single oxygen and hydrogen peroxide (Orie et al., 1999). Extracellular ROS release by PEA cells from
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normal turbot was much higher than extracellular ROS release by PEA cells from naturally infected turbot. Intracellular ROS release by PEA cells from normal turbot was markedly increased following elicitation by microsporidian spores (as opposed to thioglycollate), but no such e#ect was observed in the case of PEA cells from naturally infected turbot. Kim et al. (1998) demonstrated that spores of the microsporidian Glugea plecoglossi are capable of inhibiting the production of O 2 by head-kidney macrophages of ayu. However, in other studies performed with bacteria, increased respiratory-burst responses have been observed in fish macrophages stimulated with Aeromonas salmonicida (Chung & Secombes, 1987; Enane et al., 1993). Similar results were obtained in a study of the intracellular respiratory burst of phagocytes from head-kidneys of sea bass infected by the myxosporean parasite Sphaerospora dicentrarchi (Mun˜ oz et al., 1998, 2000), and in a study of the respiratory burst in pronephros leucocytes of rainbow trout during the acute phase of proliferative kidney disease due to an unidentified myxosporean-like parasite (Chilmonczyk & Monge, 1999). Bearing in mind that teleost phagocytic cells are involved not only in non-specific responses but also in humoral immunity (Vallejo et al., 1992), these results may at least partially explain the depression of the humoral immune response detected in some microsporidian infections (Laudan et al., 1986 a,b, 1987, 1989; Wongtavatchai et al., 1995). It has been reported that opsonised particles may provoke greater intracellular ROS production than non-opsonised particles (Secombes, 1996). Most studies of this phenomenon have used bacteria opsonised with normal serum or heat-inactivated antiserum (Waterstrat et al., 1991; Ellis, 1999). Normal turbot serum does not appear to increase the phagocytosis of sheep red blood cells (Figueras et al., 1997) or of spores of the microsporidian M. caulleryi (Leiro et al., 1996b). However, we have previously found that intracellular production of ROS by turbot spleen- and pronephros-resident adherent cells stimulated in vitro with microsporidian spores increases in the presence of normal turbot serum (Leiro et al., 1997). In addition, the opsonisation of the bacterium Renibacterium salmoninarum with normal serum from rainbow trout, or A. salmonicida with normal serum from Atlantic salmon, leads to an increase in the macrophage and neutrophil respiratory burst (Lamas & Ellis, 1994; Campos-Pérez et al., 1997). In the present study, we found that the presence of infected turbot serum decreased intracellular ROS production by PEA cells obtained from healthy turbot after elicitation with microsporidian spores. Similarly, serum from winter flounder naturally infected with Glugea stephani has likewise been shown to have an immunosuppressive e#ect, though on the humoral rather than cellular response (Laudan et al., 1986 a,b). Various hypotheses can be put forward to explain the inhibitory e#ect of turbot serum on ROS production, although the explanations for the observed e#ects on intra- and extra-cellular responses may not necessarily be the same. First, traces of haemoglobin might be present in serum, leading to catalysis of the reduction of H2O2 to OH (Sadrzadh et al., 1984). Second, serum might reduce the adherence of the spores to the PEA cells (Leiro et al., 1996b) as we found that binding of microsporidian spores to phagocytic cells occurs via direct interaction of spore-surface structures with phagocyte receptors, by a serum-independent process, possibly as a result of alteration by serum
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enzymes (e.g. chitinases) of surface molecules involved in recognition (Leiro et al., 1997). Third, net ROS release may be reduced by serum enzymes with antioxidant activity (i.e. detoxifying enzymes), such as caeruloplasmin (which converts molecular oxygen to water; Alexander & Ingram, 1992), superoxide dismutase (which converts O 2 to H2O2; Ellis, 1999) or catalase (which converts H2O2 to O2 and H2O; Ellis, 1999). Fourth, serum cytokines such as soluble macrophage-activating factor (MAF), although alone enhances O 2 production by fish macrophages (Graham & Secombes, 1988), in the presence of LPS may inhibit O 2 production, as reported for gilthead seabream macrophages (Mulero & Meseguer, 1998); similarly, interleukin-1 has immunoregulatory e#ects (Secombes et al., 1999). Finally, the observed e#ects may be due to other soluble factors such as eicosanoids (prostaglandins, prostacyclin and thromboxanes) that downregulate macrophage respiratory burst activity (Novoa et al., 1996; Tafalla et al., 1999), or hormones such as melanin-concentrating hormone (MCH) (Harris et al., 1998) or cortisol, which is capable of inhibiting the inflammatory response (Pickering & Pottinger, 1985; Narnaware et al., 1994; Espelid et al., 1996; Harris et al., 2000). In conclusion, turbot injected with microsporidian spores showed a significant increase in the number of neutrophils present in the peritoneal cavity. OxyBURST Green reagents, which fluoresce on oxidation by reactive oxygen intermediates, enabled sensitive quantification of the respiratory burst. Our results indicate that natural infection by microsporidians depresses both intra- and extra-cellular ROS production by turbot PEA cells in response to microsporidian spores. In addition, infected turbot serum appears to contain components that modulate ROS-production. This work was financially supported by grants PGIDT99MAR20301 from the Xunta de Galicia and 1FD97-0032 from the UE-FEDER/DGESIC (Ministerio de Educación y Cultura, Spain).
References Afonso, A., Lousada, S., Silva, J., Ellis, A. E. & Silva, M. T. (1998a). Neutrophil and macrophage responses to inflammation in the peritoneal cavity of rainbow trout Oncorhynchus mykiss. A light and electron microscope cytochemical study. Diseases of Aquatic Organisms 34, 27–37. Afonso, A., Silva, J., Lousada, S., Ellis, A. E. & Silva, M. T. (1998b). Uptake of neutrophils and neutrophilic components by macrophages in the inflamed peritoneal cavity of rainbow trout (Oncorhynchus mykiss). Fish & Shellfish Immunology 8, 319–338. Ainsworth, A. J. & Dexiang, C. (1990). Di#erences in the phagocytosis of four bacteria by channel catfish neutrophils. Developmental and Comparative Immunology 14, 201–209. Ainsworth, A. J., Quian, Y., Boyd, B. & Xue, L. (1996). Increased adhesion of channel catfish, Ictalurus punctatus Rafinesque, neutrophils to substrates following stimulation with phorbol ester. Fish & Shellfish Immunology 6, 35–45. Alexander, J. B. & Ingram, G. A. (1992). Noncellular nonspecific defence mechanisms of fish. Annual Review of Fish Diseases 2, 249–279. Amigó, J. M., Salvadó, H., Gracia, M. P. & Vivarés, C. P. (1996). Ultrastructure and development of Microsporidian ovoideum (Thélohan, 1895) Sprague, 1977, a microsporidian parasite of the red band fish (Cepola macrophthalma L.). European Journal of Protistology 32, 532–538.
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Mauël, J. (1997). Intracellular survival of Protozoan parasites with special reference to Leishmania spp., Toxoplasma gondii and Trypanosoma cruzi. Advances in Parasitology 38, 1–51. Modha, J., Redman, C. A., Lima, S., Kennedy, M. W. & Kusel, J. (1997). Studies of the surface properties, lipophilic proteins and metabolism of parasites by the use of fluorescent and ‘caged’ compounds. In Analytical Parasitology (M. T. Rogan, eds) pp. 269–303. Berlin: Springer-Verlag. Mulero, V. & Messeguer, J. (1998). Functional characterisation of a macrophageactivating factor produced by leucocytes of gilthead seabream (Sparus aurata L.). Fish & Shellfish Immunology 8, 143–156. Mun˜ oz, P., Sitjà-Bobadilla, A. & Alvarez-Pellitero, P. (2000). Cellular and humoral immune response of European sea bass (Dicentrarchus labrax L.) (Teleostei: Serranidae) immunized with Sphaerospora dicentrarchi (Myxosporea: Bivalvulida). Parasitology 120, 465–477. Mun˜ oz, P., Calduch-Giner, J. A., Sitjá-Bobadilla, A., Alvarez-Pellitero, P. & PérezSánchez, J. (1998). Modulation of the respiratory burst activity of Mediterranean sea bass (Dicentrarchus labrax L.) phagocytes by growth hormone and parasitic status. Fish & Shellfish Immunology 8, 25–36. Narnaware, Y. K., Baker, B. I. & Tomlinson, M. G. (1994). The e#ect of various stresses, corticosteroids and adrenergic agents on phagocytosis in the rainbow trout Oncorhynchus mykiss. Fish Physiology and Biochemistry 13, 31–40. Novoa, B., Figueras, A., Ashton, Y. & Secombes, C. J. (1996). In vitro studies on the regulation of rainbow trout (Oncorhynchus mykiss) macrophage respiratory burst activity. Developmental and Comparative Immunology 20, 207–216. Orie, N. N., Zidek, N. & Tepel, M. (1999). Tyrosine and calcium/calmodulin kinases are common signaling components in the generation of reactive oxygen species in human lymphocytes. Life Sciences 65, 2135–2140. Person-Le Ruyet, J. (1990). Sole and turbot culture. In Aquaculture, Vol. 2 (G. Barnabé, eds) pp. 667–734. Chichester: Ellis Horwood. Pickering, A. D. & Pottinger, T. G. (1985). Cortisol can increase the susceptibility of brown trout, Salmo trutta L., to disease without reducing the white blood cell count. Journal of Fish Biology 27, 611–619. Pulsford, A. & Matthews, R. A. (1991). Macrophages and giant cells associated with a microsporidian parasite causing liquefaction of the skeletal muscle of the Norway pout, Trisopterus esmarkii (Nilsson). Journal of Fish Diseases 14, 67–78. Ryan, T. C., Weil, G. J., Newburger, P. E., Haugland, R. & Simons, E. R. (1990). Measurement of superoxide release in the phagovacuoles of immune complex stimulated human neutrophils. Journal of Immunological Methods 130, 223–233. Sadrzadeh, S. M. H., Graf, E., Panter, S. S., Hallaway, P. E. & Eaton, J. W. (1984). Haemoglobin, a biological Fenton reagent. Journal of Biological Chemistry 259, 14354–14356. Schmidt, E. C. & Shadduck, J. A. (1984). Mechanisms of resistance to the intracellular protozoan Encephalitozoon cuniculi in mice. Journal of Immunology 133, 2712–2719. Secombes, C. R. (1996). The nonspecific immune system: cellular defenses. In The Fish Immune System. Organism, Pathogen, and Environment (G. Iwama & T. Nakanishi, eds), Vol. 15: Fish Physiology Series (W. S. Hoar, D. J. Randall & A. P. Farrel, eds) pp. 63–103. San Diego, CA: Academic Press. Secombes, C. J., Bird, S., Cunningham, C. & Zou, J. (1999). Interleukin-1 in fish. Fish & Shellfish Immunology 9, 335–343. Secombes, C. J. & Fletcher, T. C. (1992). The role of phagocytes in the protective mechanisms of fish. Annual Review of Fish Diseases 2, 53–71. Tafalla, C., Novoa, B. & Figueras, A. (1999). Suppressive e#ect of turbot (Scophthalmus maximus) leucocyte-derived supernatants on macrophage and lymphocyte functions. Fish & Shellfish Immunology 9, 157–166.
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Vallejo, A. W., Miller, N. W. & Clem, L. W. (1992). Antigen processing and presentation in teleost immune response. Annual Review of Fish Diseases 2, 73–89. Vossbrinck, C. R., Maddox, J. V., Friedman, B. A., Debrunner-Vossbrinck, B. A. & Woese, C. R. (1987). Ribosomal RNA sequence suggests microsporidia are extremely ancient eucaryotes. Nature 326, 411–414. Waterstrat, P. R., Ainsworth, A. J. & Capley, G. (1991). In vitro responses of channel catfish, Ictalurus punctatus, neutrophils to Edwardsiella ictaluri. Developmental and Comparative Immunology 15, 53–63. Weidner, E. (1975). Interactions between Encephalitozoon cuniculi and macrophage parasitophorous vacuole growth and the absence of lysosomal fusion. Zeitschrift für Parasitenkund 47, 1–9. Wongtavatchai, J., Conrad, P. A. & Hedrick, R. P. (1995). E#ect of the microsporidian Enterocytozoon salmonis on the immune response of chinook salmon. Veterinary Immunology and Immunopathology 48, 367–374.
Effect of Tetramicra brevifilum (Microspora) infection on respiratory-burst responses of turbot (Scophthalmus maximus L.) phagocytes J. LEIRO1*, R. IGLESIAS2, A. PARAMAu 2, M. L. SANMARTIuN2
AND
F. M. UBEIRA1
Departamento de Microbiología y Parasitología and 2Instituto de Investigación y Análisis Alimentarios, Laboratorio de Parasitología, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain 1
(Received 1 November 2000, accepted after revision 23 January 2001, published electronically 3 August 2001) In vitro assays were performed to investigate microsporidian-induced intracellular and extracellular production of reactive oxygen species (ROS) by peritoneal-exudate adherent (PEA) cells from turbot. ROS production was quantified using the fluorescent reagents OxyBURST Green H2HFF BSA (extracellular) and OxyBURST Green H2DCFDA succinimidyl ester (intracellular). Five days before assay, the cells had been elicited in vivo by intraperitoneal injection of sodium thioglycollate or spores of Tetramicra brevifilum. Elicitation with spores led to a marked increase in the proportion of neutrophils among PEA cells. PEA cells from normal turbot showed considerable extracellular and intracellular ROS production in response to microsporidian spores. By contrast, PEA cells from microsporidian-infected turbot showed considerably reduced extracellular and intracellular ROS production in response to microsporidian spores. Extracellular ROS production was a#ected by the addition of infected turbot serum to the assay medium, regardless of whether the PEA cells had been obtained from normal or infected fish. The presence of microsporidian-infected turbot serum significantly reduced intracellular ROS production by PEA cells elicited with microsporidian spores. These results suggest that (a) microsporidian spores partially suppress the repiratory-burst response of turbot phagocytes; and (b) infected turbot serum contains substances capable of modulating the respiratory-burst response of turbot phagocytes to microsporidian spores. 2001 Academic Press Key words:
turbot, microsporidians, neutrophils.
respiratory-burst,
macrophages,
I. Introduction Of the parasites capable of infecting farmed turbot, microsporidians are probably those that provoke the greatest losses (Person-Le Ruyet, 1990; Estévez et al., 1992; Figueras et al., 1992; Lom & Dyková, 1992). Microsporidians are primitive eukaryotic intracellular parasites (Vossbrinck et al., 1987). In fish, very little is known about their biology or their immunobiology (Lom & Dyková, 1992). Currently, the only species of micro*Corresponding author. E-mail: [email protected] 1050–4648/01/070639+14 $35.00/0
2001 Academic Press
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sporidian recognised as a pathogen of the turbot is Tetramicra brevifilum (Matthews & Matthews, 1980). The biology of this species within turbot has been characterised in detail (Matthews & Matthews, 1980), but the rest of its life-cycle is not known. In addition, very limited information is available on the immunobiology of turbot infected by this parasite, although there have been studies on humoral immune responses in natural infections (Leiro et al., 1993, 1996a) and of in vitro non-specific responses of turbot immune cells to microsporidian spores (Leiro et al., 1996b, 1997, 2000). During microsporidian infection in fishes, phagocytosis constitutes the final stage of defence and plays an important role in destruction of the spores (Dyková & Lom, 1980; Pulsford & Matthews, 1991; Lom & Dyková, 1992). Generally, after internalisation of the parasite by a phagocyte, it is destroyed by toxic oxygen metabolites produced during the respiratory burst (Babior, 1978; Mauël, 1982; Secombes & Fletcher, 1992). Like other intracellular protozoa, however, microsporidians may replicate within the parasitophorous vacuoles of phagocytes (Weidner, 1975; Didier, 1995). In mammals, some microsporidians are able to survive in the intracellular location, probably because of their capacity to inhibit the respiratory burst, or to avoid in other ways the e#ects of phagosome–lysosome fusion (Mauël, 1982; Schmidt & Shadduck, 1984; Didier et al., 1994; Didier, 1995). In the current study, we used a sensitive fluorescent assay to monitor intracellular and extracellular reactive oxygen species (ROS) production by adherent cells from inflamed-peritoneal exudates of normal and naturally infected turbot, and evaluated the possible modulatory e#ects of components present in the serum of normal and naturally infected turbot on the respiratory burst of phagocytes. II. Materials and Methods FISH AND SERA
Uninfected juvenile turbot Scophthalmus maximus L. (50–100 g) were obtained from a local farm (Insuin˜ a, El Grove, Pontevedra, north-western Spain). Naturally infected turbot (50–100 g) were obtained from a fish farm in the north of Galicia (north-western Spain). Prior to experiments, the fish were acclimatised for at least 15 days in 10 l tanks with a constant flow of water (181 C, pH 6·50·5) and aeration. The fish received a standard semi-dried pelleted food daily. Blood samples were collected by caudal vein puncture. The serum was separated by centrifugation at 2000g for 10 min and stored at 30 C until use.
PARASITES
Viable microsporidian spores (T. brevifilum, Matthews & Matthews, 1980) were isolated from naturally infected turbot, as described previously (Estévez et al., 1992), and stored at 4 C in sterile sea water containing 0·1% sodium
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azide. Spore viability was determined by the method of Modha et al. (1997), which is based on the incorporation of fluorescent compounds into the spore wall (Leiro et al., 2000). ELICITATION OF INFLAMMATORY CELLS
Inflammatory cells were elicited by i.p. injection of 1 ml of 3% Brewer thioglycollate medium, or 109 T. brevifilum spores suspended in PBS, into anaesthetised turbot (0·03% 2-phenoxy ethanol, Sigma), and harvested 5–6 days later. COLLECTION OF PERITONEAL LEUCOCYTES
The fish were anaesthetised as indicated above, and the anaesthesia was prolonged until death. The abdominal side of the fish was wetted with 70% ethanol, and the fish were injected through the peritoneal wall at the midline using a 10 ml syringe attached to a 22 G needle, with 5–10 ml (depending on the size of the fish) of phenol-red-free Hanks’ balanced salts solution (HBSS) containing Ca2+ and Mg2+ . Using the same syringe and needle, peritoneal fluid was then slowly withdrawn and pooled in 50 ml polypropylene centrifuge tubes on ice. PREPARATION OF PERITONEAL EXUDATE ADHERENT CELLS
Cell concentration was counted using a haemocytometer and adjusted in HBSS to 106 cells ml 1 by centrifugation at 500g for 15 min at 4 C. Aliquots of the cell suspension (100 l) were then added to the wells of 96 well microculture plates (Corning, U.S.A.) and left for 60 min at 23 C to allow adhesion. Non-adherent cells were then removed by two washes with 200 l well 1 of HBSS. The number of viable adherent cells was determined using the trypan blue exclusion test. Trypan blue (0·4% in PBS) was added to wells and incubated for 3 min at room temperature, after which the number of unstained (viable) and stained (non-viable) cells was counted. QUANTIFICATION OF EXTRACELLULAR RELEASE OF ROS
The fluorogenic reagent OxyBURST Green H2HFF BSA (Molecular Probes, The Netherlands) was used for the detection of extracellular ROS release by peritoneal-exudate adherent (PEA) cells (Ryan et al., 1990). This reagent consists of bovine serum albumin coupled to dihydro-2 ,4,5,6,7,7 hexafluorofluorescein (H2HFF), which becomes fluorescent on oxidation. BSA is a large protein, so the reagent does not cross the cell membrane, ensuring detection of extracellular oxidants only. For the assay, 100 l of PEA cells (106 cells ml 1) were incubated with microsporidian spores (cell:spore ratio 1:85, Leiro et al., 1995) for 1 h at 23 C, washed twice with HBSS and incubated in HBSS containing 10 g ml 1 of OxyBURST H2HFF Green BSA in a 96 well microculture plate. The release of oxidative products was quantified in a microplate fluorescence reader (Bio-Tek Instruments, U.K.; excitation 488 nm, detection 530 nm) as the increase in fluorescence over a 55 min period. QUANTIFICATION OF INTRACELLULAR RELEASE OF ROS
For the detection of intracellular ROS production by PEA cells, we used the amine-reactive fluorogenic reagent OxyBURST Green H2DCFDA
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succinimidyl ester (Molecular Probes, The Netherlands). Following conjugation to amines, the acetates of this reagent are removed by treatment with hydroxylamine to yield the H2DCF conjugate. As the H2DCF moiety is non-fluorescent until oxidised to dichlorofluorescein when the conjugated particles are internalised within the phagovacuole, this reagent is particularly suitable for detecting the intracellular oxidative burst in phagocytic cells (Cao et al., 1993). For microsporidian spore labelling, 100 l of the OxyBURST reagent (10 mg ml 1 in dimethylsulfoxide, DMSO) was added to a suspension of 10 mg of spores in 1 ml of 0·05 M borate bu#er pH 8·3, and incubated for 1 h in the dark at room temperature with continuous gentle shaking. To remove the acetates, 0·1 ml of 1·5 M hydroxylamine pH 8·5 was then added to the reaction mixture, which was incubated for 1 h at room temperature with stirring, then overnight at 4 C without stirring. Finally, the conjugate was separated from the unreacted reagent by repeated centrifugation in PBS at 10 000g for 3 min, until fluorescence in the supernatant dropped to zero. The spores were stored in the dark at 4 C in PBS containing 0·01% sodium azide. For the assay, 1 l aliquots of the spore suspension (previously centrifuged at 10 000g for 3 min to remove the sodium azide) were added to the wells of microculture plates containing PEA cells. Fluorescence emission was then determined in a microplate fluorescence reader (Bio-Tek Instruments; excitation 488 nm, detection 530 nm) over a 55 min period. CYTOCHEMISTRY
Peroxidase staining PEA cells on microscope slides were fixed with 10% formol-ethanol (10 ml of 37% formaldehyde and 90 ml of absolute ethanol) for 60 s at room temperature (Kaplow, 1965), then gently washed with distilled water and incubated for 15 min in TBS bu#er (50 mM Tris, 0·15 M NaCl, pH 7·4) containing 0·003% H2O2, 0·06% diaminobenzidine tetrahydrochloride and 0·03% NiCl2. The slides were then washed with TBS and contrasted with Giemsa stain for 10 min. After washing with distilled water, the slides were air-dried and permanent mounts were prepared with Eukitt. Controls were performed by omitting hydrogen peroxide from the incubation mixture. Stained cells (dark brown cytoplasm) were counted as peroxidase-positive. Non-specific esterase staining Macrophages were identified among PEA cells by determination of acid -naphthyl acetate esterase activity (ANAE staining; Ennist & Jones, 1983), as described previously (Leiro et al., 2000). DATA PRESENTATION AND STATISTICAL ANALYSIS
Intracellular formation/extracellular release of ROS were expressed in terms of variation of fluorescence (in arbitrary units) per unit of time (min). Results shown in the figures are expressed as means S.E. of three replicate
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Percentage of peritoneal– exudate adherent cells
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Fig. 1. Percentages of PEA cells showing -naphthyl acetate esterase (ANAE) activity ( ) or peroxidase activity ( ). Results are shown for cells elicited in vivo with T. brevifilum spores (A) or with sodium thioglycollate (B). There was a statistically significant di#erence between A and B (P<0·01) in the proportion of cells with peroxidase activity, but not in the proportion of cells with ANAE activity.
assays, each using the pooled adherent cells from five turbot. Significant di#erences between two means (P<0·05 or P<0·01) were determined by unpaired Student’s two-tailed t-test. The Welch t-test was used when the populations showed significant di#erence in S.D. III. Results PEROXIDASE AND NON-SPECIFIC ESTERASE ACTIVITIES OF PERITONEAL-EXUDATE ADHERENT CELLS
Peroxidase and non-specific esterase staining was used to identify cell types among the PEA cells elicited by i.p. injection, 5 days previously, of sodium thioglycollate or T. brevifilum spores. Cells showing peroxidase activity were classed as neutrophils, and cells showing nonspecific esterase activity as macrophages. Fig. 1 shows the percentages of PEA cells in each category. Nearly all cells were either macrophages or neutrophils. The proportion of neutrophils was much higher in fish injected with microsporidian spores than in fish injected with thioglycollate. EXTRACELLULAR RELEASE OF ROS BY PEA CELLS
Fig. 2 shows the results of assays of extracellular release of reactive oxygen species by PEA cells incubated with (a) microsporidian spores alone, (b) microsporidian spores plus normal turbot serum, (c) microsporidian spores plus infected turbot serum, or (d) microsporidian spores plus the ROSscavenger L(+) ascorbic acid. The PEA cells were from fish that had been injected 5 days previously with sodium thioglycollate (Fig. 2a) or T. brevifilum spores (Fig. 2b). The trypan blue exclusion test indicated that viabilities were 903% for PEA cells incubated with microsporidian spores alone, 953% for PEA cells incubated with spores plus normal turbot serum, and 925% for
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Fig. 2. Extracellular ROS production (i.e. fluorescence emission by oxidised OxyBURST Green H2HFF BSA) by PEA cells obtained from normal turbot (a) and (b) and microsporidian-infected turbot (c) and (d) after in vivo elicitation with sodium thioglycollate (a) and (c) or T. brevifilum spores (b) and (d). The PEA cells were incubated at 23 C in Hanks’ balanced salt solution with microsporidian spores ( ) and, in some experiments, 10% normal turbot serum (NTS) ( ) or microsporidianinfected turbot serum (ITS) ( ). For negative controls, 100 M L(+) ascorbic acid was included in the incubation medium (). Values shown are the means of increase of fluorescence (in arbitrary units) min1 S.E. of three replicate assays. Asterisks indicate a statistically significant di#erence (P<0·05) with respect to the corresponding value obtained after stimulation with T. brevifilum spores.
PEA cells incubated with spores plus microsporidian-infected turbot serum: in other words, none of the assay components were significantly cytotoxic. Net ROS release was markedly reduced by the inclusion of L(+) ascorbic acid in the reaction mixture, regardless of whether elicitation had been with thioglycollate or microsporidian spores (Fig. 2a and b). In assays with PEA cells elicited in vivo with thioglycollate, the addition of infected turbot serum likewise markedly reduced net ROS release (Fig. 2a). In assays of PEA cells elicited in vivo with microsporidian spores, the addition of turbot serum had no e#ect on ROS release (Fig. 2b). Fig. 2 (c and d) shows the results of assays of extracellular release of ROS by PEA cells from naturally T. brevifilum infected turbot where the PEA cells had been elicited in vivo 5 days previously with thioglycollate (Fig. 2c) or T. brevifilum spores (Fig. 2d). The fish used in this experiment were naturally infected, with large numbers of xenomas observable in the musculature, and a
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400
645
Groups
(d) 400
200 *
*
**
0 Groups
Fig. 3. Intracellular ROS production (i.e. fluorescence emission by oxidised OxyBURST Green H2DCFDA succinimidyl ester) by PEA cells obtained from normal turbot (a) and (b) or microsporidian-infected turbot (c) and (d) after in vivo elicitation with sodium thioglycollate (a) and (c) or T. brevifilum spores (b) and (d). The PEA cells were incubated with spores of T. brevilfilum ( ) and, in some experiments, 10% normal turbot serum (NTS) ( ) or microsporidian-infected turbot serum (ITS) ( ). For negative controls, 150 mM sodium azide was included in the incubation medium ( ), and without PEA cells but with spores H2DCF (). All values shown are the mean of fluorescence increase (in arbitrary units) per min S.E. after subtraction of those obtained in identical assays but without spores in the incubation medium. Asterisks indicate significant di#erences (*P<0·05; **P<0·01) with respect to the value obtained after stimulation with microsporidian spores.
mean infection intensity of 16106 spores g1 of muscle (range 2105– 4107). Extracellular ROS production in response to spores alone by PEA cells from infected fish following elicitation with thioglycollate was very low (Fig. 2c), and the response following elicitation with microsporidian spores was only slightly higher (Fig. 2d). In all cases, ROS production was reduced by the addition of infected turbot serum to the assay mixture (Fig. 2c and d). INTRACELLULAR RELEASE OF ROS BY PEA CELLS
Fig. 3 shows the results of assays of intracellular (i.e. withinphagolysosome) release of ROS by PEA cells from normal turbot (Fig. 3a and b) and T. brevifilum infected turbot (Fig. 3c and d) 5 days after elicitation with thioglycollate (Fig. 3a and c) or T. brevifilum spores (Fig. 3b or d). As in the previous experiments, PEA cells were incubated with (a) T. brevifilum spores
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alone, (b) microsporidian spores plus normal turbot serum or (d) microsporidian spores plus 1% sodium azide (negative control, no ROS production). Net ROS release by PEA cells from normal turbot was much higher after elicitation with microsporidian spores than after elicitation with thioglycollate (Fig. 3b and a, respectively). In assays with PEA cells obtained from normal turbot after elicitation with microsporidian spores, the presence of infected turbot serum led to reduced ROS release (Fig. 3b) and the presence of infected turbot serum had a significant e#ect on ROS release by PEA cells from infected turbot (Fig. 3d). IV. Discussion In fish, both macrophages and neutrophils can generate ROS (Secombes, 1996; Dalmo et al., 1997); indeed, in some species, for example Atlantic salmon, the production of O 2 following stimulation with phorbol esters is greater from neutrophils than macrophages (Lamas & Ellis, 1994). It has also been demonstrated that not only macrophages, but also neutrophils show phagocytic activity in fishes (Ainsworth & Dexiang, 1990), including the turbot (Leior et al., 2000). Furthermore, the number of neutrophils is greatly increased during inflammation induced by phlogistic agents (e.g. India ink, casein, incomplete Freund’s adjuvant, formol-killed bacteria; MacArthur et al., 1984; Hine, 1992; Afonso et al., 1998a,b), by phorbol esters (Ainsworth et al., 1996) or by microsporidian infections (Amigó et al., 1996; Leiro et al., 1999). The xenoma-production phase of microsporidian infection provokes proliferative inflammation (Lom & Dyková, 1992), characterised by the presence of fibroblasts and phagocytic cells (largely macrophages and neutrophils) (Pulsford & Matthews, 1991; Lom & Dyková, 1992; Leiro et al., 1999). In the present study, the injection of turbot with microsporidian spores greatly increased the production of neutrophils among the PEA cells. It has been demonstrated that some microsporidians can inhibit phagolysosome formation (Weidner, 1975; Schmidt & Shadduck, 1984), and that macrophages phagocytosing microsporidian spores may also secrete prostaglandins which suppress the immune response in fish (Laudan et al., 1987). To date, however, there have been no studies of the possible mechanisms by which microsporidians can escape the phagocyte respiratory burst. In principle, two possible mechanisms might be hypothesised: (i) non-elicitation of the respiratory burst (Mauël, 1997), and (ii) neutralisation of the ROS produced during the respiratory burst. In the present study, we investigated the in vitro production of ROS, in response to microsporidian spores, by PEA cells that had been elicited in vivo with either microsporidian spores or thioglycollate. Both extracellular and intracellular ROS production were investigated, using fluorogenic reagents. For negative controls in assays of extracellular ROS production, we used L(+) ascorbic acid, which is a potent scavenger of reactive oxygen and nitrogen species (Carr & Frei, 1999). For negative controls in assays of intracellular ROS production, we used sodium azide, which is a potent inhibitor of oxidative phosphorylation and catalase activity, and thus inhibits ROS release by quenching single oxygen and hydrogen peroxide (Orie et al., 1999). Extracellular ROS release by PEA cells from
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normal turbot was much higher than extracellular ROS release by PEA cells from naturally infected turbot. Intracellular ROS release by PEA cells from normal turbot was markedly increased following elicitation by microsporidian spores (as opposed to thioglycollate), but no such e#ect was observed in the case of PEA cells from naturally infected turbot. Kim et al. (1998) demonstrated that spores of the microsporidian Glugea plecoglossi are capable of inhibiting the production of O 2 by head-kidney macrophages of ayu. However, in other studies performed with bacteria, increased respiratory-burst responses have been observed in fish macrophages stimulated with Aeromonas salmonicida (Chung & Secombes, 1987; Enane et al., 1993). Similar results were obtained in a study of the intracellular respiratory burst of phagocytes from head-kidneys of sea bass infected by the myxosporean parasite Sphaerospora dicentrarchi (Mun˜ oz et al., 1998, 2000), and in a study of the respiratory burst in pronephros leucocytes of rainbow trout during the acute phase of proliferative kidney disease due to an unidentified myxosporean-like parasite (Chilmonczyk & Monge, 1999). Bearing in mind that teleost phagocytic cells are involved not only in non-specific responses but also in humoral immunity (Vallejo et al., 1992), these results may at least partially explain the depression of the humoral immune response detected in some microsporidian infections (Laudan et al., 1986 a,b, 1987, 1989; Wongtavatchai et al., 1995). It has been reported that opsonised particles may provoke greater intracellular ROS production than non-opsonised particles (Secombes, 1996). Most studies of this phenomenon have used bacteria opsonised with normal serum or heat-inactivated antiserum (Waterstrat et al., 1991; Ellis, 1999). Normal turbot serum does not appear to increase the phagocytosis of sheep red blood cells (Figueras et al., 1997) or of spores of the microsporidian M. caulleryi (Leiro et al., 1996b). However, we have previously found that intracellular production of ROS by turbot spleen- and pronephros-resident adherent cells stimulated in vitro with microsporidian spores increases in the presence of normal turbot serum (Leiro et al., 1997). In addition, the opsonisation of the bacterium Renibacterium salmoninarum with normal serum from rainbow trout, or A. salmonicida with normal serum from Atlantic salmon, leads to an increase in the macrophage and neutrophil respiratory burst (Lamas & Ellis, 1994; Campos-Pérez et al., 1997). In the present study, we found that the presence of infected turbot serum decreased intracellular ROS production by PEA cells obtained from healthy turbot after elicitation with microsporidian spores. Similarly, serum from winter flounder naturally infected with Glugea stephani has likewise been shown to have an immunosuppressive e#ect, though on the humoral rather than cellular response (Laudan et al., 1986 a,b). Various hypotheses can be put forward to explain the inhibitory e#ect of turbot serum on ROS production, although the explanations for the observed e#ects on intra- and extra-cellular responses may not necessarily be the same. First, traces of haemoglobin might be present in serum, leading to catalysis of the reduction of H2O2 to OH (Sadrzadh et al., 1984). Second, serum might reduce the adherence of the spores to the PEA cells (Leiro et al., 1996b) as we found that binding of microsporidian spores to phagocytic cells occurs via direct interaction of spore-surface structures with phagocyte receptors, by a serum-independent process, possibly as a result of alteration by serum
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enzymes (e.g. chitinases) of surface molecules involved in recognition (Leiro et al., 1997). Third, net ROS release may be reduced by serum enzymes with antioxidant activity (i.e. detoxifying enzymes), such as caeruloplasmin (which converts molecular oxygen to water; Alexander & Ingram, 1992), superoxide dismutase (which converts O 2 to H2O2; Ellis, 1999) or catalase (which converts H2O2 to O2 and H2O; Ellis, 1999). Fourth, serum cytokines such as soluble macrophage-activating factor (MAF), although alone enhances O 2 production by fish macrophages (Graham & Secombes, 1988), in the presence of LPS may inhibit O 2 production, as reported for gilthead seabream macrophages (Mulero & Meseguer, 1998); similarly, interleukin-1 has immunoregulatory e#ects (Secombes et al., 1999). Finally, the observed e#ects may be due to other soluble factors such as eicosanoids (prostaglandins, prostacyclin and thromboxanes) that downregulate macrophage respiratory burst activity (Novoa et al., 1996; Tafalla et al., 1999), or hormones such as melanin-concentrating hormone (MCH) (Harris et al., 1998) or cortisol, which is capable of inhibiting the inflammatory response (Pickering & Pottinger, 1985; Narnaware et al., 1994; Espelid et al., 1996; Harris et al., 2000). In conclusion, turbot injected with microsporidian spores showed a significant increase in the number of neutrophils present in the peritoneal cavity. OxyBURST Green reagents, which fluoresce on oxidation by reactive oxygen intermediates, enabled sensitive quantification of the respiratory burst. Our results indicate that natural infection by microsporidians depresses both intra- and extra-cellular ROS production by turbot PEA cells in response to microsporidian spores. In addition, infected turbot serum appears to contain components that modulate ROS-production. This work was financially supported by grants PGIDT99MAR20301 from the Xunta de Galicia and 1FD97-0032 from the UE-FEDER/DGESIC (Ministerio de Educación y Cultura, Spain).
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