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An in vitroassay to measure phagocytosis in striped bass hybrids (Morone saxatilis × Morone chrysops)
Fish & Shellfish Immunology (2000) 10, 405–418 doi:10.1006/fsim.1999.0248 Available online at http://www.idealibrary.com on
An in vitro assay to measure phagocytosis in striped bass hybrids (Morone saxatilisMorone chrysops) ROY E. POLONIO1*, RICHARD E. WOLKE1, SHARON A. MACLEAN2 AND JAY F. SPERRY3 1
Department of Fisheries, Animal & Veterinary Sciences, University of Rhode Island, Kingston, RI 02881, U.S.A. 2 NOAA-National Marine Fisheries Service, 28 Tarzwell Drive, Narragansett, RI 02882, U.S.A. 3 Department of Biochemistry, Microbiology and Molecular Genetics, University of Rhode Island, Kingston, RI 02881, U.S.A. (Received 3 March 1999, accepted after revision 17 November 1999) An in vitro phagocytosis assay was developed for hybrid striped bass (Morone saxatilisMorone chrysops), using cells collected from the peritoneal cavity of this fish. The findings indicated that: (1) 10 days following a single intraperitoneal injection (1 ml) of Freund’s incomplete adjuvant (FIA) was an appropriate time for collecting suitable working concentrations (5·3 4·0107 cells ml 1) of peritoneal phagocytes (83·71·5% macrophages) from these hybrids held at 23 C; (2) these cells phagocytosed latex beads (polystyrene microspheres 3·12 m in diameter) after 30 min of in vitro incubation at room temperature (251 C). The phagocytic ability and phagocytic capacity in a washed adherent layer exposure system were 67·22·76% and 4·140·35 beads phagocyte 1, respectively. These results strongly suggest that a simple methodology, including baseline data serving as guidelines, is now available for conducting in vitro phagocytosis assays in this hybrid. 2000 Academic Press Key words:
striped bass hybrid, peritoneal phagocytes, macrophages, phagocytosis, latex beads, in vitro assay.
I. Introduction Presently the culture of hybrid striped bass (Morone saxatilis Walbaum femaleMorone chrysops Rafinesque male, HSB) is of economic importance throughout the U.S.A. Many industries for commercial production of striped bass and its hybrids have emerged during the last decade in New England (NY Times, 6 November 1994), the mid-Atlantic states (Smith, 1989; van Olst & Carlberg, 1990) and to a larger extent, on the west coast. The high demands of this fish for human consumption and as fingerlings for stock enhancement in the national recreational fishery (Harrell et al., 1990) suggest an increase *Corresponding author. Mailing address: Department of Biochemistry, Microbiology and Molecular Genetics, University of Rhode Island, Kingston RI 02881, U.S.A. E-mail: rpol8053@ posto$ce.uri.edu 1050–4648/00/050405+14 $35.00/0
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in HSB aquaculture and, therefore, an increased need to understand disease prevention and control. Currently, infectious diseases are a major limiting factor in finfish aquaculture; breeders and growers of this fish have reported its susceptibility to columnaris, motile aeromonas septicemia (MAS), pasteurellosis, mycobacteriosis and vibriosis (Hawke et al., 1987; Hughes et al., 1990; Plumb, 1991). The Striped Bass Committee of the American Fisheries Society has identified rapid and e#ective disease diagnosis and control as a high priority research area (Harrell et al., 1990). The development of simple methods to assess and, if possible, modulate the hybrid’s defence against pathogens is, therefore, strongly encouraged. Phagocytosis is an essential component of the non-specific immune response against infectious agents in teleosts. This process involves the recognition and attachment of foreign particles, including pathogens, engulfment and digestion by the phagocyte. In vitro assays have been used for studying fish macrophage phagocytic activity (Weeks & Warriner, 1986; Esteban et al., 1998), thereby providing an avenue for evaluating immunocompetence in fish. In vitro assays have also provided insight for non-specifically enhancing disease resistance in finfish aquaculture (Secombes, 1994; Mulero et al., 1998), and have served as immunological biomarker tests to assess aquatic environmental health (Weeks & Warriner, 1984, 1986; Weeks et al., 1987, 1990, 1992; Dunier, 1994; Zeliko#, 1994). These assays have been developed and used in rainbow trout, Oncorhynchus mykiss (Zeliko# & Enane, 1991; Secombes, 1990), Atlantic salmon, Salmo salar (Engstad & Robertsen, 1993) and winter flounder, Pleuronectes americanus (Daniels, 1988), but this has not been done for the hybrid striped bass, Morone saxatilisMorone chrysops. As a result, the present study was conducted to develop an in vitro phagocytosis assay using peritoneal macrophages from the HSB and latex microspheres. The study focused on determining an appropriate time for collecting a suitable working concentration of peritoneal macrophages, and examining the feasibility of employing a washed adherent layer exposure system for conducting phagocytosis assays using these cells. The information provided in this study should serve as guideline and baseline data for future in vitro phagocytosis assays in this hybrid. II. Materials and Methods ACCLIMATION OF FISH
Hybrid striped bass, measuring 30·671·81 cm and weighing 724·2116·2 g (mean S.D.), were obtained from Aquafuture Inc., Turners Falls, MA, U.S.A. These hybrids, also known as sunshine, were acclimated for 4 weeks in the holding system at the Comparative Aquatic Pathology Laboratory at East Farm, University of Rhode Island, U.S.A. Initially, the fish were acclimated to 211 C for 2 weeks in a quarantine tank of the holding system equipped with biofiltration, temperature control and continuous water flow (95% reuse). The water quality was monitored daily throughout the study and was kept well within the safe levels recommended
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for this hybrid. The pertinent water quality parameters and their respective levels were: dissolved oxygen, 8–12 ppm; unionised ammonia, 0·001–0·01 ppm; nitrite–nitrogen, 0·05–0·09 ppm; nitrate–nitrogen, 5–7 ppm; water hardness (calcium carbonate/total hardness), 100/120 ppm; pH 7·1–7·5 and temperature 211 C. All fish were fed commercial striped bass feed (Moore–Clark Co. Inc., Canada) at 1% BW/D throughout the study and were kept under constant illumination (L-24) which approximates the level and duration used by Aquafuture Inc. STUDY 1: ELICITATION AND COLLECTION OF PERITONEAL CELLS
Elicitation Two weeks after initial acclimation, hybrids were divided randomly into two groups of 15, and each group was maintained for another 2 weeks; one group was kept at 23 C and the other at 15 C. After final acclimation, fish from the two temperature groups were treated to elicit peritoneal cells. This entailed anaesthetising each fish with 100 mg l 1 tricaine methanesulphonate (MS-222, bu#ered pH 7·0), measuring standard length and giving each fish an intraperitoneal (i.p.) injection (1 ml) of Freund’s incomplete adjuvant (FIA). Fish were then returned to their respective 1200 l experimental tanks for varying time periods before cell collection. Each temperature group had five subgroups of three fish which were marked by fin clipping. Cells were harvested from fish on days 1, 2, 4, 7 and 10 following i.p. injection. Prior to cell collection every fish was anaesthetised and exsanguinated. COLLECTION AND WASHING
Cell collection followed the method of Zeliko# & Enane (1991) with modification. Each fish was placed in dorsal recumbency for killing. The ventral surface was wiped with 70% ethanol and 20 ml of sterile heparinised Leibovitz’s L-15 medium (10 l heparin ml 1; Gibco, Grand Island, NY, U.S.A.) was injected into the peritoneal cavity. The abdomen was massaged gently for 1 min to facilitate collection of cells from the abdominal cavity. Following another wash with 70% ethanol, a ventral midline incision was made. Lavage fluid was drawn from each fish and transferred to sterile polypropylene tubes for washing. The peritoneal exudate cells were washed three times by centrifugation for 5 min at 770g and resuspended in fresh non-heparinised L-15. After final wash, cells were resuspended in 1 ml of L-15 for haemocytometer counting. Viability, identification and quantitation An aliquot of cells was taken from each fish sample to assess cell number and viability using a haemocytometer and trypan blue exclusion, respectively. The remaining suspension was concentrated by centrifugation and subsequent resuspension of the cell pellet in 0·5 ml L-15. Exudate smears were made,
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stained by the peroxidase/myeloperoxidase method (Sigma Diagnostics, St Louis, MO, U.S.A.), and examined by light microscopy for di#erential counting. Leucocyte di#erential counts were also performed. To ensure the stain was working properly, a negative control was employed using autologous blood smears and carrying out all but the diaminobenzidine/peroxide reagent step of the staining procedure. STUDY 2: ADHERENCE
Incubation of elicited cells Five striped bass hybrids were maintained at 23 C for 10 days following i.p. injection with FIA. Exudate cells collected from each fish were washed, counted and viability assessed as described in Study 1. This time however, 30–40 ml L-15 was delivered into the cavity and the lavage procedure was prolonged (3–5 min) to enhance cell collection. For each fish sampled, an exudate smear was made for di#erential leucocyte counting and stained by the peroxidase/myeloperoxidase method, to estimate the mean percentage of macrophages present. Cell suspension was adjusted to 1108 viable cells ml 1 in L-15 supplemented with 2% (v/v) foetal bovine serum (GBS, Gibco, Grand Island, NY, U.S.A.) and subjected to three separate incubations: 30, 90 and 180 min. For each incubation, 200 l of this sample was placed into each Lab-Tek culture well (Nunc Inc., Naperville, IL, U.S.A.) and incubated at room temperature (251·2 C) in a humidified chamber. At the end of each incubation period the cells were washed vigorously with Hanks balanced saline solution (HBSS, Gibco), removing unattached cells. Slides were air dried and methanol-fixed, and stained by the peroxidase/ myeloperoxidase method. Assessment of in vitro adherence Adherence was assessed by light microscopy, with the aid of an image analysis system (Optimas 4.1, BioScan Inc., Edmonds, WA, U.S.A.), at magnification 430. Ten fields on each microscope slide were randomly selected, and the images of those adherent cells occurring in each field were printed on to engineering forms subdivided into square inch grids (Dennison National Company, Holyoke, MA, U.S.A.). Following the standard approach used in haemocytometer counting, the number of cell images per square inch was determined in 200 randomly chosen square inch grids for each incubation period. The mean number of cell images per square inch represented on the forms served as an index of adherence, as this parameter reflected directly the number of cells per given areas on the glass slide. STUDY 3: EVALUATING PHAGOCYTOSIS OF LATEX BEADS IN EXPOSURE SYSTEM
An in vitro exposure system described herein as the washed adherent layer (WAL) system was employed. This system was modelled from phagocytosis assays involving the production of cell monolayers developed in mammals
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Table 1. Numbers of adherent peritoneal cell images per square inch at 30, 90 and 180 min incubation 30
Incubation time (min) 90
180
1·75 2·35 2·52 2·40 1·94
3·55 2·53 2·92 2·42 2·54
2·90 2·07 2·81 2·56 1·70
2·190·15a
2·790·21a
2·410·23a
Fish 1 2 3 4 5 n=5 Mean S.E.
a Analysed using a one-way ANOVA. Values are means S.E.M. Means with the same superscript are not significantly di#erent (P>0·05).
(Kavet & Brain, 1980). In the WAL system, cells were incubated with sterile latex microspheres (3·12 m polystyrene latex beads; Polysciences, Inc., Warrington, PA, U.S.A.) in culture wells of Lab-Tek glass slides. Preparation of peritoneal phagocytes and beads for incubation Each of the 10 fish was maintained at 23 C for 10 days following a single i.p. injection of FIA. Cells collected from each fish were washed, counted and viability assessed as described in Study 2. An exudate smear was also made for each fish sampled, and stained by the peroxidase/myeloperoxidase method, to determine the mean percentage of macrophages present. The cell suspension was adjusted to 1107 viable cells ml 1 in L-15 supplemented with 2% (v/v) FBS. The bead suspension was diluted with L-15 medium and adjusted to 1108 beads ml 1; this concentration was used in each phagocytosis trial. WASHED ADHERENT LAYER (WAL)
Aliquots (200 l) of cell suspension (1107 cells ml 1) from each fish were placed into culture wells and incubated for 30 min at room temperature (25·01·5 C). Thirty minutes was the selected incubation time because Study 2 showed that this period was adequate for very good attachment and spreading of phagocytes on glass surfaces at room temperature (Table 1). At the end of this attachment period, cells were washed three times with HBSS. This was immediately followed by the addition of 200 l of bead suspension to the adherent layer of phagocytes in each well. The adherent cells were then co-incubated with latex beads for 30 min and at the end of this period, cells were washed with HBSS. SLIDE PREPARATION AND LIGHT MICROSCOPIC ANALYSES
All slides were air dried, methanol fixed and then treated with xylene as demonstrated by van Furth & Diesselholf-den Dulk (1980) to dissolve any extracellular beads, thereby increasing significantly the degree of accuracy
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Table 2. Mean number (107 cells ml 1) of elicited peritoneal cells on days 1, 2, 4, 7 and 10 from hybrid striped bass held at 15 and 23 C Day 1 2 4 7 10
15
Temperature ( C)
1·60·8 1·90·8 1·90·8 2·81·8 1·00·5
23
1·91·3 0·60·2 4·21·4 4·82·8 5·34·0
Data are expressed as mean valuesstandard error of the mean (S.E.M.); n=3 for each day samples.
in quantitating phagocytosis. The cells were again air dried and stained by the peroxidase/myeloperoxidase method. Slides were examined by light microscopy 430 with the aid of an image analysis system (Optimas 4.1). The phagocytic ability, described by Esteban et al. (1998), was evaluated by estimating the mean percentage phagocytes containing at least one bead, in a random count of 200 cells. The phagocytic capacity was evaluated by estimating the mean number of beads/phagocyte in a random count of 100 cells that had internalised microspheres. STATISTICAL EVALUATION
Results were expressed as the mean... The data from Study 2 was analysed with Statmost for Windows software package (DATAMost Corporation, Salt Lake City, UT, U.S.A.), using a one-way analysis of variance (ANOVA). Di#erences between the mean values for adherence were considered significant at P<0·05. III. Results ELICITATION, VIABILITY AND CELL NUMBER
Light microscope examination (100) of haemocytometer-mounted peritoneal fluid revealed peritoneal leucocytes as very refractile and somewhat spherical to slightly irregular in shape, most having rough edges. Cell viability was above 95% in every sample. Cell collection was greatest (5·34·0107 viable cells ml 1) on day 10 in fish held at 23 C (Table 2). Cell recovery from fish held at 15 C was generally much lower, the highest being 2·81·0107 viable cells ml 1 on day 7. IDENTIFICATION AND QUANTITATION OF ELICITED PERITONEAL CELLS
The neutrophils of this fish were di#erentially stained by the peroxidase/ myeloperoxidase method, thus enabling the identification of di#erent leucocyte types collected from the peritoneal cavity. Neutrophils had large
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Fig. 1. Photomicrograph (brightfield) showing peroxidase/myeloperoxidase staining reactions of peritoneal cells. The peroxidase-positive neutrophils (N) contain dense, dark granules whereas the peroxidase-negative macrophages (M) are lacking this characteristic. Scale bar=20 m.
cytoplasmic granules that stained dark brown, while both the lymphocytes and macrophages lacked this characteristic (Fig. 1). The latter two cell types had azurophilic cytoplasm and a densely stained basophilic nucleus. Macrophages had an eccentric nucleus and a much larger cytoplasm/nucleus ratio than lymphocytes; the latter generally possessed a large nucleus surrounded by a thin cytoplasmic layer. Eosinophils were extremely rare and had large, densely stained red, cytoplasmic granules. Basophils were not found. The relative percentage of peritoneal macrophages varied with number of days after injection (Fig. 2). There was a general increase in number of macrophages with time at both temperature regimes, but this occurred at a slower rate in the 15 C group. The highest macrophage populations (83·71·5%) were obtained on day 10 in the 23 C group. The percentage of elicited peritoneal neutrophils declined steadily over time (Fig. 2). Neutrophils were included in this report only for the purpose of illustrating the relative proportions of these two cell types during the course of an inflammatory response, such as that elicited by FIA. ADHERENCE OF ELICITED CELLS
The corresponding di#erential exudate smears revealed that an average of 862·0% of the cells used in this study were peroxidase negative. Cell viability was consistently above 95%. The cells that adhered to the glass slide of the chamber wells following washing were peroxidase-negative and markedly pleomorphic. (Few of these cells were weakly peroxidase positive). Some appeared somewhat rounded and others spindle shaped, spreading extensively on glass surfaces, with poorly stained cytoplasmic processes extending in various directions. These phagocytes ranged in sizes from 9·25 to 42·82 m at their longest axes.
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100 90 Mean percentage ± S.E.
80 70
15° C 23° C
60 50 40 30 20 10 0
23° C 15° C 1
2 3 4 5 6 7 8 9 Days following i.p. injection (1 ml FIA)
10
11
Fig. 2. Mean percentage of elicited peritoneal macrophages (squares) and neutrophils (triangles) collected on days 1, 2, 4, 7 and 10 from fish held at 15 C (open symbols) and 23 C (closed symbols), n=3, for each day sampled.
Adherence, as indexed by the number of cell images per square inch, was greatest with 2·790·21 images per square inch after 90 min, followed by 2·410·23 and 2·190·15 images per square inch after 180 and 30 min, respectively (Table 1). However, analysis of data from the three incubations revealed that no statistically significant di#erences existed in adherence of phagocytes with respect to incubation time (P>0·05). EVALUATION OF IN VITRO PHAGOCYTOSIS OF LATEX BEADS IN WAL
The corresponding di#erential exudate smears revealed that an average of 861·4% of the cells used in this study were peroxidase negative. Cell viability was consistently above 95%. These phagocytes contained beads and exhibited identical features to those seen in Study 2 (Fig. 3). The phagocytic ability and phagocytic capacity were 67·22·76% and 4·140·35 bead/ phagocyte, respectively (Table 3). Cells were somewhat evenly distributed on the glass surface. IV. Discussion ELICITATION AND CELL DENSITY
Elicitation was greatest on day 10 at 23 C. This time was, therefore, considered appropriate for collecting suitable numbers of peritoneal cells, of which 84% were macrophages. Similar results (85%) have been reported for channel catfish, Ictalurus punctatus, that were injected i.p. with 0·5 ml FIA and squalene to elicit peritoneal macrophages (Jenkins & Leedy, 1994).
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Fig. 3. Photomicrograph (brightfield) showing hybrid striped bass macrophages laden with beads (arrows) in the washed adherent layer (WAL) exposure system. Stain: peroxidase/myeloperoxidase. Scale bar=10 m.
Table 3. Phagocytosis of beads by peritoneal macrophages in Washed Adherent Layer exposure system Fish 1 2 3 4 5 6 7 8 9 10 Mean SE
Phagocytic ability*
Phagocytic capacity**
75·0 65·0 48·5 60·5 75·0 63·0 72·0 66·0 77·5 70·0 67·22·76
5·43 3·74 2·99 2·32 5·22 4·59 4·16 2·97 5·18 4·79 4·140·35
*Phagocytic ability is the percentage of 200 randomly counted macrophages that had ingested beads. **Phagocytic capacity is the mean number of beads ingested by 200 macrophages that had ingested beads.
Generally, during an inflamatory response in mammals and fish, the neutrophils arrive at the site of infection first while monocytes and macrophages come in later and persist (Suzuki & Iida, 1992). This pattern was demonstrated by peritoneal cells in this study (Fig. 2). A lower temperature regime was introduced in this study for comparison and to observe the e#ect of decreased environmental temperature on elicitation. It is believed that the non-specific immune response of certain teleosts
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is markedly depressed at 15 C (Collazos et al., 1994). The data obtained in this study from fish held at 15 C supports this hypothesis and agrees with the findings reported for trout by Finn & Nielsen (1971), showing that decreased environmental temperature delayed inflammatory responses of these poikilotherms. The large standard errors accompanying the cell numbers may be attributed to intraspecific variations among fish and, perhaps to inherent limitations in the collection method. Insu$cient lavage fluid was recovered from the peritoneal cavity of some bass, especially those with excess visceral adipose tissue. Further, the delivery into the peritoneal cavity of only 20 ml lavage fluid may have contributed to some of the unexpectedly low cell numbers and thus, the variations. These variations were nonetheless greatly reduced during studies 2 and 3, in which the volume of fluid delivered into the peritoneal cavity was increased to 30–40 ml and the lavage period was prolonged (3–5 min). These modifications promoted the collection of consistently high working concentrations (3·30·6107 viable cells ml 1) of macrophage-enriched peritoneal cells, 86% (1·4) of which were indicative of macrophages (data not shown). IDENTIFICATION
As morphology provided little or no help in di#erentiating elicited peritoneal cells, including granulocytes, macrophage/monocytes and lymphocytes, enzyme cytochemistry was employed. The strong peroxidase activity observed in neutrophils of this hybrid was consistent with reports on neutrophil peroxidase reaction in striped bass, Morone saxatilis (Bodammer et al., 1990), channel catfish, Ictalurus punctatus (Cannon et al., 1980), plaice Pleuronectes platessa L. (Ellis, 1976) and rainbow trout, Oncorhynchus mykiss (Ainsworth, 1992). The negative peroxidase reaction demonstrated by the macrophages in this study is also similar to the findings of Zeliko# & Enane (1991) in rainbow trout and Calduch-Giner et al. (1997) in gilthead seabream, Sparatus aurata. Therefore, monocytes/macrophages of this fish are probably poor or lacking in peroxidase enzymes which may have only very little involvement in the antibacterial functions of these phagocytes. The inverse applies to the neutrophils; this hypothesis however requires experimental confirmation. The weak peroxidase reaction occurring in a few cells may have resulted from some transfer to macrophages of neutrophilic peroxidase, as reported by Afonso et al. (1998). It is possible however, that these cells were indeed neutrophils, showing only weak reaction to the myeloperoxidase stain. But, to determine conclusively whether this is the case is beyond the scope of this study. In studies 2 and 3, peritoneal cells were collected from these fish 10 days following elicitation and were essentially peroxidase-negative. Therefore, while recognising the potential involvement of neutrophils, the time of cell collection and the peroxidase/myeloperoxidase staining characteristics strongly suggests that the phagocytes used in the development of this assay were primarily macrophages.
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ADHERENCE AND WAL EXPOSURE SYSTEM
Cells that adhered to the glass surface and internalised beads were usually peroxidase negative. These phagocytes attached very well after 30 min of incubation, producing adherent layers. In vitro phagocytosis assays developed in other fish species have often incubated cells for various attachment periods, ranging between 60 min and 3 h (Weeks & Warinner, 1984; Secombes, 1990; Zeliko# & Enane, 1991; Engstad & Robertsen, 1993; Jørgensen et al., 1993; Browser et al., 1994). This study, however, has clearly shown that for HSB peritoneal phagocytes, 30 min was adequate for attachment and therefore, for the production of adherent layers on glass surfaces. Many reviews have been published (Kavet & Brain, 1980; Schroeder & Kidan, 1983; Fernandez-Repollet et al., 1988) on phagocytosis assays, including those dealing with cell suspensions. Phagocytosis in a WAL system occurs exclusively while cells are attached to glass surface; this conclusion cannot be made for cell suspension assay systems. The WAL system also takes into account attachment of these cells to substratum when assessing phagocyte functions; cell suspension assay systems do not account for such crucial behaviour in these cells. These cells, like their mammalian counterparts, were phagocytic; and demonstrated a remarkable ability to attach and spread on glass surfaces. This behaviour also suggested that most of these elicited cells were activated. Macrophage activation is a phenomenon that has been described in mammalian cells and in salmonid macrophages (Karnovsky et al., 1975; Adams & Hamilton, 1984; Chung & Secombes, 1987; Zeliko# & Enane, 1991). Activated macrophages are described as being large in size, having pronounced cell membrane ru%ing, enhanced capacity for adherence and spreading on solid surfaces and enhanced phagocytic and microbicidal ability (Cohn, 1978; North, 1978; Nathan, 1986). The di#erences in degrees of spreading, phagocytic capacity and cell sizes among macrophages may be attributed to di#erences in the level of activation and/or the presence of di#erent macrophage subpopulations (Zeliko# & Enane, 1991). The variations in the degrees of spreading of the phagocytes observed in the present study warrant further investigation. The adherent cells of the HSB were very similar in appearance to mammalian macrophages, both when loaded with beads and when lacking beads. In this exposure system, the phagocytes and the internalised beads were easily quantified. A limitation, however, was the unknown number of adherent cells following the first 30 min of attachment period. This short-coming, however, would only a#ect phagocytosis assays requiring exact numbers of cells. Estimation and standardisation of macrophage adherence are useful, and have been performed in mammalian in vitro systems, showing that 50–70% of these elicited cells remained attached to the glass surfaces (Hercowitz & Cole, 1981). The development of a similar protocol for the HSB is recommended. CONCLUSION
This study has determined that: (1) enrichment for peritoneal macrophages is attainable in the hybrid bass; (2) these elicited cells can easily be identified,
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quantified and maintained in vitro; and (3) these phagocytes, in a WAL system, can adhere to glass surfaces and actively take up latex beads. The study therefore concludes that a simple and inexpensive methodology, including baseline data serving as guidelines, is now available for studying in vitro phagocytosis by peritoneal macrophages in this hybrid. This work is part of a thesis, submitted in partial fulfilment of the requirements for the Master of Science degree in Fisheries, Aquaculture and Pathology, University of Rhode Island, Kingston Rhode Island. We are grateful to the College of Resource Development of the University of Rhode Island, contribution #: 3480, and to the Fulbright scholarship program, Institute of International Education (IIE), for supporting this research. We would also like to thank Paul W. Johnson for developing the photomicrographs herein, Sheila Polofsky for her moral support and to Drs David Bengtson and Joel Bodammer for their constructive criticism of the original manuscript.
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Environmental Toxicology Biomarkers Immunostimulators (J. S. Stolen, T. C. Fletcher, D. P. Anderson, J. T. Zeliko#, L. E. Twerdok, S. L. Kaattari & C. C. Bayne, eds) pp. 123–139. Fair Haven, NJ: SOS Publications. Ellis, A. E. (1976). Leukocytes and related cells in the plaice Pleuronectes platessa. Journal of Fish Biology 8, 143–156. Engstad, R. E. & Robertson, B. (1993). Recognition of yeast cell wall glycan by Atlantic salmon Salmo salar, macrophages. Developmental and Comparative Immunology 17, 319–330. Esteban, M. A. & Meseguer, J. (1997). Factors influencing phagocytic response of macrophages from the sea bass (Dicentrarchus labrax L.) an ultrastructural and quantitative study. The Anatomical Record 248, 533–541. Esteban, M. A., Mulero, V., Munoz, J. & Meseguer, J. (1998). Methodological aspects of assessing phagocytosis of Vibrio anguillarium by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy. Cell Tissue Research 293, 133–141. Fernandez-Repollet, E. & Schwartz, A. (1988). Microspheres and phagocytosis. In Microspheres Medical and Biological Applications (A. Rembaum & Z. A. Tokes, eds) pp. 139–164. Boca Raton, FL: CRC Press Inc. Finn, J. P. & Nielsen, N. O. (1971). The e#ect of temperature variation on inflammatory response of rainbow trout. Journal of Pathology 105, 257–268. Harrell, R. M., Kerby, J. H., Smith, T. I. J. & Stevens, R. E. (1990). Striped bass and striped bass hybrid culture: The next twenty-five years. In Culture and Propagation of Striped Bass and its Hybrids (R. M. Harrell, J. H. Kerby & R. V. Milton, eds) pp. 253–262. Bethesda, MD: American Fisheries Society. Hawke, J. P., Plakas, S. M., Minton, R. V., McPherson, R. M., Snider, T. G. & Guarino, A. M. (1987). Fish pasteurellosis of cultured striped bass Morone saxatilis in coastal Alabama. Aquaculture 65, 193–204. Hercowitz, H. B. & Cole, D. E. (1981). Maintenance of macrophages in vitro. In Manual of Macrophage Methodology; Collection, Characterization and Function (H. B. Hercowitz, H. T. Holdei, J. A. Bellanti & A. Gha#ai, eds) pp. 161–169. NY: Maicel Pekkei, Inc. Hughes, J. S., Wellbourne, T. L. & Mitchell, A. J. (1990). Parasites and diseases of striped bass and hybrids. In Culture and Propagation of Striped Bass and its Hybrids (R. M. Harrell, J. H. Kerby & R. V. Minton, eds) pp. 217–238. Bethesda, MD: American Fisheries Society. Jenkins, J. A. & Leedy, D. H. (1994). Optimized elicitation of macrophages from the peritoneal cavity of channel catfish (Ictalurus punctatus). In Fased Journal, Experimental Biology 95. Vol. 9 No. 3 (W. J. Whelan, ed.) p. 522. Atlanta, Georgia. Jørgensen, J. B., Sharp, G. J. E., Secombes, C. J. & Robertsen, B. (1993). E#ect of a yeast-cell-wall glucan on the bactericidal activity of rainbow trout macrophages. Fish & Shellfish Immunology 3, 267–277. Karnovsky, M. L., Lazdins, J., Drath, D. & Harper, A. (1975). Biochemical characteristics of activated macrophages. Annals of the New York Academy of Sciences 256, 266–270. Kavet, R. I. & Brain, J. D. (1980). Methods to quantify endocytosis: a review. Journal of the Reticuloendothelial Society 27, 201–221. Mulero, V., Esteban, M. A., Munoz, J. & Meseguer, J. (1998). Dietary intake of levamisole enhances the immune response and disease resistance of the marine teleost gilthead seabream. Fish & Shellfish Immunology 1, 49–62. Nathan, C. F. (1986). Macrophage activation: some questions. Annales de Institut Pasteur Immunologie 137, 345–351. North, R. J. (1978). The concept of the activated macrophage. Journal of Immunology 121, 806–809. Plumb, J. A. (1991). Major diseases of striped bass and redfish. Veterinary and Human Toxicology 33 (supplement), 34–39.
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Rabinovitch, M. & DeStafano, M. J. (1973). Macrophage spreading in vitro. Experimental Cell Research 77, 323–334. Schroeder, F. & Kinden, D. A. (1983). Measurement of phagocytosis using fluorescent latex beads. Journal of Biochemical and Biophysical Methods 89, 150–156. Secombes, C. J. (1990). Isolation of salmonid macrophages and analysis of their killing activity. In Techniques in Fish Immunology (J. S. Stolen, T. C. Fletcher, D. P. Anderson, B. S. Robertson & W. B. van Muiswinkel, eds) pp. 137–154. Fair Haven, NJ: SOS Publications. Secombes, C. J. (1994). Enhancement of fish phagocyte activity. Fish & Shellfish Immunology 4, 421–436. Smith, T. I. J. (1989). The culture potential of striped bass and its hybrids. World Aquaculture 20, 32–38. Suzuki, Y. & Iida, T. (1992). Fish granulocytes in the process of inflammation. Annual Review of Fish Diseases 2, 149–160. vanFurth, R. & Diesselhol#-den Dulk, M. M. C. (1980). Method to prove ingestion of particles by macrophages with light microscopy. Scandinavian Journal of Immunology 12, 265–269. Van Olst, J. C. & Carlberg, J. M. (1990). Commercial culture of hybrid striped bass: status and potential. Aquaculture Magazine 16, 49–59. Weeks, B. A. & Warinner, J. E. (1984). E#ects of toxic chemicals on macrophage phagocytosis in two estuarine fishes. Marine Environmental Research 14, 327– 335. Weeks, B. A. & Warinner, J. E. (1986). Functional evaluation of macrophages in fish from a polluted estuary. Veterinary Immunology and Immunopathology 12, 313–320. Weeks, B. A., Keisler, A. S., Warinner, J. E. & Mathews, E. S. (1987). Preliminary evaluation of macrophage pinocytosis as a technique to monitor fish health. Marine Environmental Research 22, 205–213. Weeks, B. A., Hugget, R. J. & Hargis, W. J. Jr (1990). Integrated chemical pathological and immunological studies to assess environmental contamination. In In situ Evaluations of Biological Hazards of Environmental Pollutants (Sandhu, ed) pp. 233–240. NY: Plenum Press. Weeks, B. A., Anderson, D. P., DuFour, A. P., Fairbrother, A., Goven, A. J., Lahvis, G. P. & Peters, G. (1992). Immunological biomarkers to assess environmental stress. In Biomarkers: Biochemical, Physiological, and Histopathological Markers of Anthropogenic Stress (R. J. Huggett, R. A. Kimerle, P. M. Mehrle & H. L. Bergman, eds) pp. 211–234. Chelsea, MI: Lewis Publishers. Zeliko#, J. T. (1994). Immunological alterations as indicators of environmental metal exposure. In Modulators of Fish Immune Response: Models for Environmental Toxicology Biomarkers Immunostimulators, Vol. I (J. S. Stolen, T. C. Fletcher, D. P. Anderson, J. T. Zeliko#, L. E. Twerdok, S. L. Kaattari & C. C. Bayne, eds) pp. 101–110. Fair Haven, NJ: SOS Publications. Zeliko#, J. T. & Enane, N. A. (1991). Assays used to assess the activation state of rainbow trout peritoneal macrophages. In Techniques in Fish Immunology (J. S. Stolen, T. C. Fletcher, D. P. Anderson, S. L. Kaattari & A. F. Rowley, eds) pp. 107–124. Fair Haven, NJ: SOS Publications.
An in vitro assay to measure phagocytosis in striped bass hybrids (Morone saxatilisMorone chrysops) ROY E. POLONIO1*, RICHARD E. WOLKE1, SHARON A. MACLEAN2 AND JAY F. SPERRY3 1
Department of Fisheries, Animal & Veterinary Sciences, University of Rhode Island, Kingston, RI 02881, U.S.A. 2 NOAA-National Marine Fisheries Service, 28 Tarzwell Drive, Narragansett, RI 02882, U.S.A. 3 Department of Biochemistry, Microbiology and Molecular Genetics, University of Rhode Island, Kingston, RI 02881, U.S.A. (Received 3 March 1999, accepted after revision 17 November 1999) An in vitro phagocytosis assay was developed for hybrid striped bass (Morone saxatilisMorone chrysops), using cells collected from the peritoneal cavity of this fish. The findings indicated that: (1) 10 days following a single intraperitoneal injection (1 ml) of Freund’s incomplete adjuvant (FIA) was an appropriate time for collecting suitable working concentrations (5·3 4·0107 cells ml 1) of peritoneal phagocytes (83·71·5% macrophages) from these hybrids held at 23 C; (2) these cells phagocytosed latex beads (polystyrene microspheres 3·12 m in diameter) after 30 min of in vitro incubation at room temperature (251 C). The phagocytic ability and phagocytic capacity in a washed adherent layer exposure system were 67·22·76% and 4·140·35 beads phagocyte 1, respectively. These results strongly suggest that a simple methodology, including baseline data serving as guidelines, is now available for conducting in vitro phagocytosis assays in this hybrid. 2000 Academic Press Key words:
striped bass hybrid, peritoneal phagocytes, macrophages, phagocytosis, latex beads, in vitro assay.
I. Introduction Presently the culture of hybrid striped bass (Morone saxatilis Walbaum femaleMorone chrysops Rafinesque male, HSB) is of economic importance throughout the U.S.A. Many industries for commercial production of striped bass and its hybrids have emerged during the last decade in New England (NY Times, 6 November 1994), the mid-Atlantic states (Smith, 1989; van Olst & Carlberg, 1990) and to a larger extent, on the west coast. The high demands of this fish for human consumption and as fingerlings for stock enhancement in the national recreational fishery (Harrell et al., 1990) suggest an increase *Corresponding author. Mailing address: Department of Biochemistry, Microbiology and Molecular Genetics, University of Rhode Island, Kingston RI 02881, U.S.A. E-mail: rpol8053@ posto$ce.uri.edu 1050–4648/00/050405+14 $35.00/0
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in HSB aquaculture and, therefore, an increased need to understand disease prevention and control. Currently, infectious diseases are a major limiting factor in finfish aquaculture; breeders and growers of this fish have reported its susceptibility to columnaris, motile aeromonas septicemia (MAS), pasteurellosis, mycobacteriosis and vibriosis (Hawke et al., 1987; Hughes et al., 1990; Plumb, 1991). The Striped Bass Committee of the American Fisheries Society has identified rapid and e#ective disease diagnosis and control as a high priority research area (Harrell et al., 1990). The development of simple methods to assess and, if possible, modulate the hybrid’s defence against pathogens is, therefore, strongly encouraged. Phagocytosis is an essential component of the non-specific immune response against infectious agents in teleosts. This process involves the recognition and attachment of foreign particles, including pathogens, engulfment and digestion by the phagocyte. In vitro assays have been used for studying fish macrophage phagocytic activity (Weeks & Warriner, 1986; Esteban et al., 1998), thereby providing an avenue for evaluating immunocompetence in fish. In vitro assays have also provided insight for non-specifically enhancing disease resistance in finfish aquaculture (Secombes, 1994; Mulero et al., 1998), and have served as immunological biomarker tests to assess aquatic environmental health (Weeks & Warriner, 1984, 1986; Weeks et al., 1987, 1990, 1992; Dunier, 1994; Zeliko#, 1994). These assays have been developed and used in rainbow trout, Oncorhynchus mykiss (Zeliko# & Enane, 1991; Secombes, 1990), Atlantic salmon, Salmo salar (Engstad & Robertsen, 1993) and winter flounder, Pleuronectes americanus (Daniels, 1988), but this has not been done for the hybrid striped bass, Morone saxatilisMorone chrysops. As a result, the present study was conducted to develop an in vitro phagocytosis assay using peritoneal macrophages from the HSB and latex microspheres. The study focused on determining an appropriate time for collecting a suitable working concentration of peritoneal macrophages, and examining the feasibility of employing a washed adherent layer exposure system for conducting phagocytosis assays using these cells. The information provided in this study should serve as guideline and baseline data for future in vitro phagocytosis assays in this hybrid. II. Materials and Methods ACCLIMATION OF FISH
Hybrid striped bass, measuring 30·671·81 cm and weighing 724·2116·2 g (mean S.D.), were obtained from Aquafuture Inc., Turners Falls, MA, U.S.A. These hybrids, also known as sunshine, were acclimated for 4 weeks in the holding system at the Comparative Aquatic Pathology Laboratory at East Farm, University of Rhode Island, U.S.A. Initially, the fish were acclimated to 211 C for 2 weeks in a quarantine tank of the holding system equipped with biofiltration, temperature control and continuous water flow (95% reuse). The water quality was monitored daily throughout the study and was kept well within the safe levels recommended
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for this hybrid. The pertinent water quality parameters and their respective levels were: dissolved oxygen, 8–12 ppm; unionised ammonia, 0·001–0·01 ppm; nitrite–nitrogen, 0·05–0·09 ppm; nitrate–nitrogen, 5–7 ppm; water hardness (calcium carbonate/total hardness), 100/120 ppm; pH 7·1–7·5 and temperature 211 C. All fish were fed commercial striped bass feed (Moore–Clark Co. Inc., Canada) at 1% BW/D throughout the study and were kept under constant illumination (L-24) which approximates the level and duration used by Aquafuture Inc. STUDY 1: ELICITATION AND COLLECTION OF PERITONEAL CELLS
Elicitation Two weeks after initial acclimation, hybrids were divided randomly into two groups of 15, and each group was maintained for another 2 weeks; one group was kept at 23 C and the other at 15 C. After final acclimation, fish from the two temperature groups were treated to elicit peritoneal cells. This entailed anaesthetising each fish with 100 mg l 1 tricaine methanesulphonate (MS-222, bu#ered pH 7·0), measuring standard length and giving each fish an intraperitoneal (i.p.) injection (1 ml) of Freund’s incomplete adjuvant (FIA). Fish were then returned to their respective 1200 l experimental tanks for varying time periods before cell collection. Each temperature group had five subgroups of three fish which were marked by fin clipping. Cells were harvested from fish on days 1, 2, 4, 7 and 10 following i.p. injection. Prior to cell collection every fish was anaesthetised and exsanguinated. COLLECTION AND WASHING
Cell collection followed the method of Zeliko# & Enane (1991) with modification. Each fish was placed in dorsal recumbency for killing. The ventral surface was wiped with 70% ethanol and 20 ml of sterile heparinised Leibovitz’s L-15 medium (10 l heparin ml 1; Gibco, Grand Island, NY, U.S.A.) was injected into the peritoneal cavity. The abdomen was massaged gently for 1 min to facilitate collection of cells from the abdominal cavity. Following another wash with 70% ethanol, a ventral midline incision was made. Lavage fluid was drawn from each fish and transferred to sterile polypropylene tubes for washing. The peritoneal exudate cells were washed three times by centrifugation for 5 min at 770g and resuspended in fresh non-heparinised L-15. After final wash, cells were resuspended in 1 ml of L-15 for haemocytometer counting. Viability, identification and quantitation An aliquot of cells was taken from each fish sample to assess cell number and viability using a haemocytometer and trypan blue exclusion, respectively. The remaining suspension was concentrated by centrifugation and subsequent resuspension of the cell pellet in 0·5 ml L-15. Exudate smears were made,
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stained by the peroxidase/myeloperoxidase method (Sigma Diagnostics, St Louis, MO, U.S.A.), and examined by light microscopy for di#erential counting. Leucocyte di#erential counts were also performed. To ensure the stain was working properly, a negative control was employed using autologous blood smears and carrying out all but the diaminobenzidine/peroxide reagent step of the staining procedure. STUDY 2: ADHERENCE
Incubation of elicited cells Five striped bass hybrids were maintained at 23 C for 10 days following i.p. injection with FIA. Exudate cells collected from each fish were washed, counted and viability assessed as described in Study 1. This time however, 30–40 ml L-15 was delivered into the cavity and the lavage procedure was prolonged (3–5 min) to enhance cell collection. For each fish sampled, an exudate smear was made for di#erential leucocyte counting and stained by the peroxidase/myeloperoxidase method, to estimate the mean percentage of macrophages present. Cell suspension was adjusted to 1108 viable cells ml 1 in L-15 supplemented with 2% (v/v) foetal bovine serum (GBS, Gibco, Grand Island, NY, U.S.A.) and subjected to three separate incubations: 30, 90 and 180 min. For each incubation, 200 l of this sample was placed into each Lab-Tek culture well (Nunc Inc., Naperville, IL, U.S.A.) and incubated at room temperature (251·2 C) in a humidified chamber. At the end of each incubation period the cells were washed vigorously with Hanks balanced saline solution (HBSS, Gibco), removing unattached cells. Slides were air dried and methanol-fixed, and stained by the peroxidase/ myeloperoxidase method. Assessment of in vitro adherence Adherence was assessed by light microscopy, with the aid of an image analysis system (Optimas 4.1, BioScan Inc., Edmonds, WA, U.S.A.), at magnification 430. Ten fields on each microscope slide were randomly selected, and the images of those adherent cells occurring in each field were printed on to engineering forms subdivided into square inch grids (Dennison National Company, Holyoke, MA, U.S.A.). Following the standard approach used in haemocytometer counting, the number of cell images per square inch was determined in 200 randomly chosen square inch grids for each incubation period. The mean number of cell images per square inch represented on the forms served as an index of adherence, as this parameter reflected directly the number of cells per given areas on the glass slide. STUDY 3: EVALUATING PHAGOCYTOSIS OF LATEX BEADS IN EXPOSURE SYSTEM
An in vitro exposure system described herein as the washed adherent layer (WAL) system was employed. This system was modelled from phagocytosis assays involving the production of cell monolayers developed in mammals
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Table 1. Numbers of adherent peritoneal cell images per square inch at 30, 90 and 180 min incubation 30
Incubation time (min) 90
180
1·75 2·35 2·52 2·40 1·94
3·55 2·53 2·92 2·42 2·54
2·90 2·07 2·81 2·56 1·70
2·190·15a
2·790·21a
2·410·23a
Fish 1 2 3 4 5 n=5 Mean S.E.
a Analysed using a one-way ANOVA. Values are means S.E.M. Means with the same superscript are not significantly di#erent (P>0·05).
(Kavet & Brain, 1980). In the WAL system, cells were incubated with sterile latex microspheres (3·12 m polystyrene latex beads; Polysciences, Inc., Warrington, PA, U.S.A.) in culture wells of Lab-Tek glass slides. Preparation of peritoneal phagocytes and beads for incubation Each of the 10 fish was maintained at 23 C for 10 days following a single i.p. injection of FIA. Cells collected from each fish were washed, counted and viability assessed as described in Study 2. An exudate smear was also made for each fish sampled, and stained by the peroxidase/myeloperoxidase method, to determine the mean percentage of macrophages present. The cell suspension was adjusted to 1107 viable cells ml 1 in L-15 supplemented with 2% (v/v) FBS. The bead suspension was diluted with L-15 medium and adjusted to 1108 beads ml 1; this concentration was used in each phagocytosis trial. WASHED ADHERENT LAYER (WAL)
Aliquots (200 l) of cell suspension (1107 cells ml 1) from each fish were placed into culture wells and incubated for 30 min at room temperature (25·01·5 C). Thirty minutes was the selected incubation time because Study 2 showed that this period was adequate for very good attachment and spreading of phagocytes on glass surfaces at room temperature (Table 1). At the end of this attachment period, cells were washed three times with HBSS. This was immediately followed by the addition of 200 l of bead suspension to the adherent layer of phagocytes in each well. The adherent cells were then co-incubated with latex beads for 30 min and at the end of this period, cells were washed with HBSS. SLIDE PREPARATION AND LIGHT MICROSCOPIC ANALYSES
All slides were air dried, methanol fixed and then treated with xylene as demonstrated by van Furth & Diesselholf-den Dulk (1980) to dissolve any extracellular beads, thereby increasing significantly the degree of accuracy
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Table 2. Mean number (107 cells ml 1) of elicited peritoneal cells on days 1, 2, 4, 7 and 10 from hybrid striped bass held at 15 and 23 C Day 1 2 4 7 10
15
Temperature ( C)
1·60·8 1·90·8 1·90·8 2·81·8 1·00·5
23
1·91·3 0·60·2 4·21·4 4·82·8 5·34·0
Data are expressed as mean valuesstandard error of the mean (S.E.M.); n=3 for each day samples.
in quantitating phagocytosis. The cells were again air dried and stained by the peroxidase/myeloperoxidase method. Slides were examined by light microscopy 430 with the aid of an image analysis system (Optimas 4.1). The phagocytic ability, described by Esteban et al. (1998), was evaluated by estimating the mean percentage phagocytes containing at least one bead, in a random count of 200 cells. The phagocytic capacity was evaluated by estimating the mean number of beads/phagocyte in a random count of 100 cells that had internalised microspheres. STATISTICAL EVALUATION
Results were expressed as the mean... The data from Study 2 was analysed with Statmost for Windows software package (DATAMost Corporation, Salt Lake City, UT, U.S.A.), using a one-way analysis of variance (ANOVA). Di#erences between the mean values for adherence were considered significant at P<0·05. III. Results ELICITATION, VIABILITY AND CELL NUMBER
Light microscope examination (100) of haemocytometer-mounted peritoneal fluid revealed peritoneal leucocytes as very refractile and somewhat spherical to slightly irregular in shape, most having rough edges. Cell viability was above 95% in every sample. Cell collection was greatest (5·34·0107 viable cells ml 1) on day 10 in fish held at 23 C (Table 2). Cell recovery from fish held at 15 C was generally much lower, the highest being 2·81·0107 viable cells ml 1 on day 7. IDENTIFICATION AND QUANTITATION OF ELICITED PERITONEAL CELLS
The neutrophils of this fish were di#erentially stained by the peroxidase/ myeloperoxidase method, thus enabling the identification of di#erent leucocyte types collected from the peritoneal cavity. Neutrophils had large
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Fig. 1. Photomicrograph (brightfield) showing peroxidase/myeloperoxidase staining reactions of peritoneal cells. The peroxidase-positive neutrophils (N) contain dense, dark granules whereas the peroxidase-negative macrophages (M) are lacking this characteristic. Scale bar=20 m.
cytoplasmic granules that stained dark brown, while both the lymphocytes and macrophages lacked this characteristic (Fig. 1). The latter two cell types had azurophilic cytoplasm and a densely stained basophilic nucleus. Macrophages had an eccentric nucleus and a much larger cytoplasm/nucleus ratio than lymphocytes; the latter generally possessed a large nucleus surrounded by a thin cytoplasmic layer. Eosinophils were extremely rare and had large, densely stained red, cytoplasmic granules. Basophils were not found. The relative percentage of peritoneal macrophages varied with number of days after injection (Fig. 2). There was a general increase in number of macrophages with time at both temperature regimes, but this occurred at a slower rate in the 15 C group. The highest macrophage populations (83·71·5%) were obtained on day 10 in the 23 C group. The percentage of elicited peritoneal neutrophils declined steadily over time (Fig. 2). Neutrophils were included in this report only for the purpose of illustrating the relative proportions of these two cell types during the course of an inflammatory response, such as that elicited by FIA. ADHERENCE OF ELICITED CELLS
The corresponding di#erential exudate smears revealed that an average of 862·0% of the cells used in this study were peroxidase negative. Cell viability was consistently above 95%. The cells that adhered to the glass slide of the chamber wells following washing were peroxidase-negative and markedly pleomorphic. (Few of these cells were weakly peroxidase positive). Some appeared somewhat rounded and others spindle shaped, spreading extensively on glass surfaces, with poorly stained cytoplasmic processes extending in various directions. These phagocytes ranged in sizes from 9·25 to 42·82 m at their longest axes.
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100 90 Mean percentage ± S.E.
80 70
15° C 23° C
60 50 40 30 20 10 0
23° C 15° C 1
2 3 4 5 6 7 8 9 Days following i.p. injection (1 ml FIA)
10
11
Fig. 2. Mean percentage of elicited peritoneal macrophages (squares) and neutrophils (triangles) collected on days 1, 2, 4, 7 and 10 from fish held at 15 C (open symbols) and 23 C (closed symbols), n=3, for each day sampled.
Adherence, as indexed by the number of cell images per square inch, was greatest with 2·790·21 images per square inch after 90 min, followed by 2·410·23 and 2·190·15 images per square inch after 180 and 30 min, respectively (Table 1). However, analysis of data from the three incubations revealed that no statistically significant di#erences existed in adherence of phagocytes with respect to incubation time (P>0·05). EVALUATION OF IN VITRO PHAGOCYTOSIS OF LATEX BEADS IN WAL
The corresponding di#erential exudate smears revealed that an average of 861·4% of the cells used in this study were peroxidase negative. Cell viability was consistently above 95%. These phagocytes contained beads and exhibited identical features to those seen in Study 2 (Fig. 3). The phagocytic ability and phagocytic capacity were 67·22·76% and 4·140·35 bead/ phagocyte, respectively (Table 3). Cells were somewhat evenly distributed on the glass surface. IV. Discussion ELICITATION AND CELL DENSITY
Elicitation was greatest on day 10 at 23 C. This time was, therefore, considered appropriate for collecting suitable numbers of peritoneal cells, of which 84% were macrophages. Similar results (85%) have been reported for channel catfish, Ictalurus punctatus, that were injected i.p. with 0·5 ml FIA and squalene to elicit peritoneal macrophages (Jenkins & Leedy, 1994).
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Fig. 3. Photomicrograph (brightfield) showing hybrid striped bass macrophages laden with beads (arrows) in the washed adherent layer (WAL) exposure system. Stain: peroxidase/myeloperoxidase. Scale bar=10 m.
Table 3. Phagocytosis of beads by peritoneal macrophages in Washed Adherent Layer exposure system Fish 1 2 3 4 5 6 7 8 9 10 Mean SE
Phagocytic ability*
Phagocytic capacity**
75·0 65·0 48·5 60·5 75·0 63·0 72·0 66·0 77·5 70·0 67·22·76
5·43 3·74 2·99 2·32 5·22 4·59 4·16 2·97 5·18 4·79 4·140·35
*Phagocytic ability is the percentage of 200 randomly counted macrophages that had ingested beads. **Phagocytic capacity is the mean number of beads ingested by 200 macrophages that had ingested beads.
Generally, during an inflamatory response in mammals and fish, the neutrophils arrive at the site of infection first while monocytes and macrophages come in later and persist (Suzuki & Iida, 1992). This pattern was demonstrated by peritoneal cells in this study (Fig. 2). A lower temperature regime was introduced in this study for comparison and to observe the e#ect of decreased environmental temperature on elicitation. It is believed that the non-specific immune response of certain teleosts
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is markedly depressed at 15 C (Collazos et al., 1994). The data obtained in this study from fish held at 15 C supports this hypothesis and agrees with the findings reported for trout by Finn & Nielsen (1971), showing that decreased environmental temperature delayed inflammatory responses of these poikilotherms. The large standard errors accompanying the cell numbers may be attributed to intraspecific variations among fish and, perhaps to inherent limitations in the collection method. Insu$cient lavage fluid was recovered from the peritoneal cavity of some bass, especially those with excess visceral adipose tissue. Further, the delivery into the peritoneal cavity of only 20 ml lavage fluid may have contributed to some of the unexpectedly low cell numbers and thus, the variations. These variations were nonetheless greatly reduced during studies 2 and 3, in which the volume of fluid delivered into the peritoneal cavity was increased to 30–40 ml and the lavage period was prolonged (3–5 min). These modifications promoted the collection of consistently high working concentrations (3·30·6107 viable cells ml 1) of macrophage-enriched peritoneal cells, 86% (1·4) of which were indicative of macrophages (data not shown). IDENTIFICATION
As morphology provided little or no help in di#erentiating elicited peritoneal cells, including granulocytes, macrophage/monocytes and lymphocytes, enzyme cytochemistry was employed. The strong peroxidase activity observed in neutrophils of this hybrid was consistent with reports on neutrophil peroxidase reaction in striped bass, Morone saxatilis (Bodammer et al., 1990), channel catfish, Ictalurus punctatus (Cannon et al., 1980), plaice Pleuronectes platessa L. (Ellis, 1976) and rainbow trout, Oncorhynchus mykiss (Ainsworth, 1992). The negative peroxidase reaction demonstrated by the macrophages in this study is also similar to the findings of Zeliko# & Enane (1991) in rainbow trout and Calduch-Giner et al. (1997) in gilthead seabream, Sparatus aurata. Therefore, monocytes/macrophages of this fish are probably poor or lacking in peroxidase enzymes which may have only very little involvement in the antibacterial functions of these phagocytes. The inverse applies to the neutrophils; this hypothesis however requires experimental confirmation. The weak peroxidase reaction occurring in a few cells may have resulted from some transfer to macrophages of neutrophilic peroxidase, as reported by Afonso et al. (1998). It is possible however, that these cells were indeed neutrophils, showing only weak reaction to the myeloperoxidase stain. But, to determine conclusively whether this is the case is beyond the scope of this study. In studies 2 and 3, peritoneal cells were collected from these fish 10 days following elicitation and were essentially peroxidase-negative. Therefore, while recognising the potential involvement of neutrophils, the time of cell collection and the peroxidase/myeloperoxidase staining characteristics strongly suggests that the phagocytes used in the development of this assay were primarily macrophages.
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ADHERENCE AND WAL EXPOSURE SYSTEM
Cells that adhered to the glass surface and internalised beads were usually peroxidase negative. These phagocytes attached very well after 30 min of incubation, producing adherent layers. In vitro phagocytosis assays developed in other fish species have often incubated cells for various attachment periods, ranging between 60 min and 3 h (Weeks & Warinner, 1984; Secombes, 1990; Zeliko# & Enane, 1991; Engstad & Robertsen, 1993; Jørgensen et al., 1993; Browser et al., 1994). This study, however, has clearly shown that for HSB peritoneal phagocytes, 30 min was adequate for attachment and therefore, for the production of adherent layers on glass surfaces. Many reviews have been published (Kavet & Brain, 1980; Schroeder & Kidan, 1983; Fernandez-Repollet et al., 1988) on phagocytosis assays, including those dealing with cell suspensions. Phagocytosis in a WAL system occurs exclusively while cells are attached to glass surface; this conclusion cannot be made for cell suspension assay systems. The WAL system also takes into account attachment of these cells to substratum when assessing phagocyte functions; cell suspension assay systems do not account for such crucial behaviour in these cells. These cells, like their mammalian counterparts, were phagocytic; and demonstrated a remarkable ability to attach and spread on glass surfaces. This behaviour also suggested that most of these elicited cells were activated. Macrophage activation is a phenomenon that has been described in mammalian cells and in salmonid macrophages (Karnovsky et al., 1975; Adams & Hamilton, 1984; Chung & Secombes, 1987; Zeliko# & Enane, 1991). Activated macrophages are described as being large in size, having pronounced cell membrane ru%ing, enhanced capacity for adherence and spreading on solid surfaces and enhanced phagocytic and microbicidal ability (Cohn, 1978; North, 1978; Nathan, 1986). The di#erences in degrees of spreading, phagocytic capacity and cell sizes among macrophages may be attributed to di#erences in the level of activation and/or the presence of di#erent macrophage subpopulations (Zeliko# & Enane, 1991). The variations in the degrees of spreading of the phagocytes observed in the present study warrant further investigation. The adherent cells of the HSB were very similar in appearance to mammalian macrophages, both when loaded with beads and when lacking beads. In this exposure system, the phagocytes and the internalised beads were easily quantified. A limitation, however, was the unknown number of adherent cells following the first 30 min of attachment period. This short-coming, however, would only a#ect phagocytosis assays requiring exact numbers of cells. Estimation and standardisation of macrophage adherence are useful, and have been performed in mammalian in vitro systems, showing that 50–70% of these elicited cells remained attached to the glass surfaces (Hercowitz & Cole, 1981). The development of a similar protocol for the HSB is recommended. CONCLUSION
This study has determined that: (1) enrichment for peritoneal macrophages is attainable in the hybrid bass; (2) these elicited cells can easily be identified,
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quantified and maintained in vitro; and (3) these phagocytes, in a WAL system, can adhere to glass surfaces and actively take up latex beads. The study therefore concludes that a simple and inexpensive methodology, including baseline data serving as guidelines, is now available for studying in vitro phagocytosis by peritoneal macrophages in this hybrid. This work is part of a thesis, submitted in partial fulfilment of the requirements for the Master of Science degree in Fisheries, Aquaculture and Pathology, University of Rhode Island, Kingston Rhode Island. We are grateful to the College of Resource Development of the University of Rhode Island, contribution #: 3480, and to the Fulbright scholarship program, Institute of International Education (IIE), for supporting this research. We would also like to thank Paul W. Johnson for developing the photomicrographs herein, Sheila Polofsky for her moral support and to Drs David Bengtson and Joel Bodammer for their constructive criticism of the original manuscript.
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