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AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY V . 12, pp. 57-71, 2006 Vol Immune function assays in Murray Cod Harford et al THE OPTIMISATION OF IMMUNE FUNCTION ASSAYS IN MURRAY COD, MACCULLOCHELLA PEELII PEELII Andrew J. Harforda, Kathryn O’Halloranb and Paul F. A. Wrighta* a Key Centre for Toxicology, School of Medical Sciences, RMIT-University, PO Box 71, Plenty Road, Bundoora, Victoria, 3083, Australia. b Manaaki Whenua Landcare Research, PO Box 40, Lincoln 7640, New Zealand. Manuscript received, 15/2/2006; accepted 27/7/2006 ABSTRACT Murray Cod (Maccullochella peelii peelii) is the largest and best-known native Australian freshwater fish, with high economic, recreational and ecological value. The Murray Cod aquaculture industry continues to be developed and has great potential, however the immunology of this species has not been studied to date. In addition, many aquatic pollutants that contaminate the habitat of Murray Cod have known immunotoxicity in both mammals and exotic fish species. In this study, two immune function assays were optimised in Murray Cod, i.e. phagocytosis measured by flow cytometry and mitogenstimulated lymphoproliferation. Based on these studies, standardised protocols are defined for the use of these assays in the assessment of immunomodulation by both environmental pollutants, as well as immunostimulants that may be applied in the aquaculture of Murray Cod. Murray Cod adapted well to laboratory conditions and are an ideal freshwater Australian species for ecoimmunotoxicology testing. Key words: Murray Cod; immunotoxicology; mitogenesis; lymphoproliferation; flow cytometry. INTRODUCTION Murray Cod (Maccullochella peelii peelii Mitchell) is the largest and best-known native Australian freshwater species and is indigenous to the Murray-Darling river system. However, its distribution and abundance has declined in the past 50 years due to the construction of dams, changes to river flows and temperatures, and increased siltation of waterways. The species is now fragmented and in low abundance and has been listed under the Commonwealth Environmental Protection & Biodiversity Conservation Act as “endangered” and a species of national significance (DEH 2005) In contrast, Murray Cod aquaculture is a promising industry with a large potential to grow rapidly. The industry now produces both fingerlings for the stocking of waterways and table-sized fish (500 to 800 g) for human consumption. In 2001, in excess of 70 tonnes of Murray Cod was produced for human consumption, which was approximately double the total production from the commercial capture fisheries from the whole of the Murray-Darling basin. In that year, the Murray Cod market was estimated to be worth $A2 million and its value continues to grow (Love and Langenkamp 2003). In 2004, the production of Murray Cod by the largest producer of native freshwater fish (Australian Aquaculture Products [AAP], Euroa, Victoria, Australia) reached over 100 tonnes (Roger Camm, AAP, pers. commun.). Research programs involving Murray Cod have focused on aquaculture solutions and the ecology of the species. The aquaculture research has included studies concerning the optimisation of spawning, rearing and dietary conditions, while ecological studies have investigated behavioural patterns and habitat requirements (Kearney and Kildea 2001). However, the immunology and immunotoxic responses of Murray Cod have not been studied to date. *Author for correspondence, email: [email protected] The immune system serves to protect the host from infectious diseases and developing neoplastic cells and is highly conserved across all vertebrate species, with remnants also existing in invertebrates (Roitt et al. 1998). It is also highly sensitive to insult from chemical exposures and many drugs have been used to modulate various aspects of the immune response (Dean and Murray 2001). Numerous assays have been developed to measure the activity of its various components, such as the innate, humoral and cellmediated immune functions, while the integrity of the entire system can be assessed through disease challenge models. The identification of immunotoxins has been achieved via a three-tiered system, which was developed and validated in mammals by the National Toxicology Program in the U.S.A (Luster et al. 1988). Studies that have applied these methods have demonstrated that many pollutants that contaminate the Murray-Darling basin are immunotoxic in mammals and exotic species of fish; however, no studies have applied standardised immune functional assays to assess the immunotoxicity of environmental pollutants in native Australian freshwater fish. Phagocytosis is a primitive defence mechanism, conserved in both vertebrates and invertebrates. This function is the first step in a cascade of events that include the destruction of the invading organism, antigen processing and presentation, and the regulation of the immune response through the secretion of cytokines (Neumann et al. 2001). The phagocytic function of fish has been used in many studies as an indicator of their health status (Secombes 1994; Secombes and Fletcher 1992) and it has also been used in a tiered system for the immunotoxicological assessment of environmental pollutants and immunostimulants used in aquaculture (Secombes 1994; Siwicki et al. 1998). Our group has recently reported the use 57 AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Immune function assays in Murray Cod of flow cytometry to measure the phagocytic activity of head kidney cells from three native Australian freshwater fish, i.e. crimson-spotted rainbowfish (Melanotaenia fluviatilis), silver perch (Bidyanus bidyanus) and golden perch (Macquaria ambigua) (Harford et al. 2006). Flow cytometry utilises the light scattering properties of cells to categorise them into different subpopulations based on their size, granularity and/or their emission of fluorescent signals and can be used to measure the internalisation of fluorescent beads by phagocytic cells (Thuvander et al. 1992). The lymphoproliferative response to mitogens is similar to the adaptive lymphocytic response to antigens that are presented by macrophages, however it does not require the action of antigen presenting cells and occurs rapidly in response to natural agents conserved in many foreign organisms, such as bacteria (e.g. lipopolysaccharide, LPS) and plants (e.g. phytohemagglutinin, PHA). It is commonly measured through the amount of radiolabelled thymidine that is incorporated into the DNA of proliferating lymphocytes. Lymphocyte mitogenesis is an immune function that has been observed in many vertebrates, including numerous fish species (Zelikoff 1998). Previous research by our laboratory has measured mitogenesis in various native Australian fish including the marine species, sand flathead (O’Halloran 1996), and freshwater species such as crimson-spotted rainbowfish (Barry et al. 1995) and silver perch (O’Halloran et al. 1996). The present study aimed to optimise two important tier 2 immune function tests (i.e. lymphoproliferation and phagocytic activity) for use in the assessment of immunomodulatory chemicals in Murray Cod. These assays are more suited for developing simple screening tests in native freshwater species than the more complex tier 3 tests, which require special pathogen containment facilities for conducting host resistance testing and are not available in most facilities. Furthermore, the use of flow cytometry also enables the quantification of immune cell subpopulations, which is a sensitive tier 1 parameter. METHODS Wet-laboratory water All fish were held in the wet-laboratory facility at RMIT. The laboratory was a flow-through design that was supplied with carbon-filtered water heated to 19 to 20°C. The water had an oxygen concentration of 7.6 to 7.8 mg/L, a pH of 6.8 to 7.2 and a conductivity of 100 to 120 μS/cm. Fish maintenance Murray Cod (150 to 250 g) were purchased from Australian Aquaculture Products (Euroa, Vic, Australia) and transported for 2 h in an aerated 1 000 L transportable tank on the back of a utility vehicle. Individual fish were then transferred to 40-L tanks receiving aeration and a constant supply (5 L/h) of fresh carbon filtered aquaria water at 20 ± 1°C. They were fed a commercially available pellet every second day (Australian Native Fish Pellet, Skretting Australia, Cambridge, Tasmania, Australia) and tanks were cleaned on the days between feeding. They were acclimatised in tanks for at least four 58 V . 12, pp. 57-71, 2006 Vol Harford et al weeks before conducting experiments. Fish that appeared stressed (i.e. immobile, not feeding and/or discoloured) or injured were not used in the studies. Cell isolation Head kidney cells were isolated using modified protocols previously described by O’Halloran et al. (1998). Briefly, cells were disrupted from the head kidney tissue and passed through a 250-μm nylon mesh. Red blood cells were separated from the cell suspension by density gradient centrifugation. The immune cells were washed twice in tissue culture media (i.e. TCM, consisting of RPMI 1640 with 20 mM N-(2hydro xyethyl)piperazine-N-2-ethane sulfonic acid (HEPES), 300 mg/L glutamine and 100 μg/mL gentamycin sulfate (Sigma Chemical Co., St Louis, MO, USA); supplemented with 10% heat-treated fetal calf serum (FCS) (CSL, Melbourne, Vic, Australia), counted using a haemocytometer in the presence of trypan blue (0.2% w/v) and diluted to the concentration required for the assays. Mitogenesis Teleost species appear to require a wide range of optimal culture conditions for the measurement of mitogenesis (Rosenberg-Wiser and Avtalian 1982; DeKoning and Kaattari 1991). Variables such as mitogen concentration, incubation time, temperature, tritium exposure time and cell concentration were investigated to formulate a standardised protocol for use in subsequent immunotoxicity studies. Mitogens Mitogens are biological agents capable of stimulating the proliferation of lymphocytes that have had no prior exposure to the agent. Since many fish species appear to respond differently to mitogens, the lymphoproliferative effects to four different mitogens were investigated in this study, i.e. PHA and two other plant lectins, pokeweed mitogen (PWM) and Concanavalin A (ConA), as well as lipopolysaccharide (LPS), a bacterial cell wall constituent. To characterise the lymphoproliferative responses to these four mitogens, the following range of concentrations were investigated; PHA 0, 0.156, 0.312, 0.625, 1.25, 2.5, 5 and 10 μg/mL; ConA - 0, 0.312, 0.625, 1.25, 2.5, 5, 10 and 20 μg/mL; LPS - 0, 0.625, 1.25, 2.5, 5, 10, 20 and 40 μg/mL and PWM - 0, 0.78, 0.156, 0.312, 0.625, 1.25, 2.5 and 5 μg/mL. Incubation temperature and duration Both the duration of exposure and the incubation temperature can influence the peak proliferative responses of fish lymphocytes to mitogens. Murray Cod head kidney cells (2.5x106 cells/mL) were incubated in 96 well plates (with the aforementioned mitogen concentrations), for 3, 5 and 7 d at 15, 20 and 25°C in TCM, with an atmosphere of 95% air/ 5% CO2. Cells were pulsed with tritiated thymidine (18.5 GBq/well, Amersham International, Amersham, UK) 24 h prior to cell harvesting using a semi-automatic 12 well harvester (Skatron, Lier, Norway). Tritiated thymidine incorporation into newly divided cells (i.e. proliferation) was quantified using a liquid scintillation counter (LKB, Wallac, Turku, Finland) and scintillation fluid (ACSII, Amersham International, Amersham, UK).. AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Immune function assays in Murray Cod Cell concentration and tritiated thymidine exposure period Murray Cod head kidney cells (1, 2.5, 5 and 10x106 cells/ mL) were incubated with PHA (0 to 10 μg/mL) for 5 d at 15°C, in order to determine the cell concentration that would produce an optimal mitogenic response. As mitogen-induced proliferation in fish cells is likely to occur at a slower rate than in mammalian cells, due to the lower incubation temperatures, it was also investigated whether adding tritiated thymidine at an earlier stage would enhance the measurement of newly divided cells. For these experiments cells were pulsed with tritiated thymidine at 48 h or 24 h prior to cell harvesting. Media conditions Osmolarity The serum osmolarity of many fish species is not the same as humans or rodents (i.e. 290 to 300 mOsm) for which most cell culture products have been optimised. Consequently, some researchers have employed media with modified osmolarity to further optimise the responses of fish lymphocytes (Scapigliati et al. 2002). The mitogenic response of Murray Cod head kidney cells was investigated at various osmolarities, with the aim of further increasing the lymphocyte response. Murray Cod head kidney cells (5.0x106 cells/mL) were cultured in TCM at various osmolarities of 190, 220, 250, 280, 310, 340 and 360 mOsm, i.e. TCM with an osmolarity of 280 mOsm was either diluted with water or the osmolarity was increased by the addition of 30 mM NaCl. Cells (5.0x106 cells/mL) were then incubated with PHA for 5 d at 15°C and were pulsed with tritiated thymidine (18.5 GBq/well) 24 h prior to harvesting. The osmolarity of Murray Cod serum was also measured with a cryoscopic osmometer (Osmomat 030, Gonotec, Berlin, Germany). Serum The use of homologous serum supplements, rather than the FCS normally used for many cell culture systems, has been reported to increase the mitogenic responses of trout (DeKoning and Kaattari 1991) and has been used to aid in the mitogenesis of carp and catfish lymphocytes (RosenbergWiser and Avtalion 1982; Faulmann 1983). Therefore, the use of homologous serum for optimising the mitogen-stimulated proliferation of lymphocytes isolated from Murray Cod head kidneys was investigated. Murray Cod head kidney cells (5.0x106 cells/mL) were cultured in either TCM with 10% FCS, no serum, 10% fresh Murray Cod serum or in 10% heat-treated Murray Cod serum. Cells (5.0x106 cells/mL) were then incubated with PHA for 5 d at 15°C and were pulsed with tritiated thymidine (18.5 GBq/well) 24 h prior to harvesting. Mercaptoethanol concentration Researchers have reported that the addition of the sulfhydryl antioxidant, mercaptoethanol (2-ME), increases the mitogenesis of fish lymphocytes. However, the concentrations used may vary between studies and fish species (RosebergWiser and Avtalion 1982; Arkoosh et al. 1994; O’Halloran 1996). A concentration range of 2-ME was investigated to determine the optimum concentration for the lymphocyte V . 12, pp. 57-71, 2006 Vol Harford et al proliferative assay. Murray Cod head kidney cells (5.0x106 cells/mL) were cultured in TCM with various concentrations of 2-ME (i.e. 0, 25, 50 and 100 μM). Cells were then incubated with PHA for 5 d at 15°C and were pulsed with tritiated thymidine (18.5 GBq/well) 24 h prior to harvesting. HEPES The addition of HEPES to the RPMI-1640 media aids in buffering the media, particularly during manipulations outside of the sealed incubation chamber. However, the addition of HEPES can also lead to the production of cytotoxic products such as hydrogen peroxide (Bowman et al. 1985; Zigler et al. 1985). Therefore, the effect of removing HEPES before culturing lymphocytes was investigated, with the aim of increasing the lymphoproliferative response. Standard cell isolation methods described in the section above were employed, except that HEPES free media was used for the washing steps after density gradient centrifugation. Murray Cod head kidney cells (5.0x106 cells/mL) were cultured in TCM either with or without HEPES, and the cells were incubated for 5 d at 15°C and pulsed with tritiated thymidine (18.5 GBq/well) 24 h prior to harvesting. Flow cytometry Phagocytic activity was quantified by the flow cytometer (EPICS Elite II, Coulter, Hialeah, FL, USA) after incubation with fluorescent (fluorescein isothiocyanate, FITC) latex beads (Polysciences Inc., Warrington, PA, USA), as previously described by Harford et al. (2006). Briefly, cells that engulfed FITC beads had a peak light emission at 520 nm (measured in PMT2), while the majority of unengulfed beads were excluded from the analysis by the utilisation of the “live gating” option available in the Epics Elite Expo32 software (Coulter, Hialeah, FL, USA). This live gating option enables the researcher to analyse events only relating to the cells, without the need for physical separation of unengulfed beads from the cell suspension prior to flow cytometry. However, there is a certain amount of background fluorescence (~10%) caused by unengulfed beads passing the laser in close proximity to cells. This background effect was corrected for during experiments by the use of a negative control, which was an identical set of samples that was incubated under the same conditions as the samples but without beads. Beads were added immediately before analysis on the flow cytometer (i.e. t = 0), and thus represented a sample where no phagocytosis had occurred. A typical flow cytometric display is shown in Figure 1. The phagocytosis data collected in this assay was expressed as the percentage of granulocytes emitting fluorescence at 520 nm (i.e. calculated as the number of FITC positive granulocyte-gated events / total granulocyte-gated events x 100, minus the negative control). The analysis of mean voltage output of FITC positive events, i.e. the intensity of the fluorescent signal, which is incrementally increased as increasing numbers of beads pass the laser together, provided an indication of the number of beads engulfed per cell. Cell viability was monitored through a change in the cell structure (i.e. events in the debris-gated region) and by the exclusion of the fluorescent vital dye propridium iodide 59 AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY V . 12, pp. 57-71, 2006 Vol Immune function assays in Murray Cod Harford et al Figure 1. The FITC “signal” measured in PMT 2 shows c) negative control with a single peak representing unengulfed beads and d) multiple peaks after incubation with beads, representing phagocytes that have internalised multiple beads. In the two-dimensional plots cell events are shown as grey dots and the FITC+ve bead events are black dots. (PI). The majority of PI fluorescent events were found in the “debris-gated” region, which exhibited low forward scatter (i.e. smaller in size) and high side scatter (i.e. a more granular cytoplasm) characteristics due to the unviable cells losing their structural integrity shortly after the membrane becomes permeable to PI. Incubation temperature and duration Murray Cod head kidney cells (1x10 6 cells/mL) were incubated in 1 mL of TCM with 2.5x107 FITC latex beads at 15, 20 and 25°C. In order to determine the optimum time for phagocytic activity, samples were analysed on days 2, 3, 5 and 7 using the flow cytometer. 60 Cell:Bead ratio Beads were not physically separated from the samples before analysis, however, the majority of beads were excluded via the “live gating” option in the Epics Elite Expo32 software. This can contribute to the background level of phagocytic events as cells that pass the laser in close proximity to a bead will result in a false positive signal. Also interference from the co-incident unengulfed beads can alter the light scattering properties of a particular event and can result in a reduction of the event counts in the various subpopulationgated regions. Both the background effect and the reduction in subpopulation counts increase with an increasing cell: bead ratio. While this effect would have been reduced by the introduction of an additional sample work-up step to remove AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Immune function assays in Murray Cod unengulfed beads from the cells, the more desirable option is to avoid further cell-processing steps by selecting a cell:bead ratio where an optimal compromise has been found between measuring the maximal phagocytic response and preventing interference in counting the immune cell subpopulations. Therefore, Murray Cod head kidney cells (1x106 cells/mL) in 1 mL of TCM were incubated for 2, 3, 5 and 7 d at 15oC with FITC latex beads at four different cell:bead ratios (1:6, 1:12.5, 1:25 and 1:50). Media conditions Osmolarity and Serum Murray Cod head kidney cells (1.0x106 cells/mL) were cultured with FITC–latex beads (1:25 cells/bead) in TCM with the same osmolarities or with the various serum supplements described in section above describing media conditions. The cell cultures were then incubated for 48 h and phagocytosis and subpopulation counts were measured on the flow cytometer. Validation of optimised conditions and polyethylene glycol as a vehicle The head kidneys of eight Murray Cod were sampled and eight additional fish received 2 mL/kg polyethylene glycol (PEG, Unilab, Sydney, New South Wales, Australia). The fish were administered a single intraperitoneal injection of PEG and their immune tissues were sampled 14 days later. The head kidney cells were incubated under the optimised conditions for mitogenesis and phagocytosis described previously. Statistical analyses Statistics were performed using the computer package SPSS 11.0 (SPSS Inc., Chicago, Illinois, USA). Analyses were performed on raw data that was first checked for normal distribution using the Kolmogorov-Smirnov test. Analysis of variance (i.e. one-way ANOVA) was performed and Tukey’s compromise post hoc test was conducted to determine homogenous subsets. A P value of less than 0.05 was considered to indicate a statistically significant difference. RESULTS Mitogenesis The effectiveness of the four different mitogens in stimulating lymphoproliferation of Murray Cod head kidney cells is illustrated in Figure 2. Of the four mitogens tested, PHA exposure resulted in the highest proliferation, with twice basal proliferation occurring at 1.25 μg/mL. Results from subsequent experiments shown in the figures indicated that the peak mitogenesis often occurred at higher PHA concentrations and that using a range of PHA concentrations in mitogenesis assessments allowed peak mitogenesis to be observed despite this variability. Of the incubation temperatures tested, 15°C appeared to be optimal for Murray Cod lymphoproliferation (Figure 3a, b and c) and the incubation period yielding greatest sensitivity was 5 d (Figure 3b). Although the mitogenic response from the fish used in the cell concentration optimisation experiment was low, increasing the cell concentration resulted in a proportional increase V . 12, pp. 57-71, 2006 Vol Harford et al with both the peak and basal counts per minute (Figure 4). Therefore, 5x106 cells/mL was chosen as the most appropriate cell concentration to provide a readily measurable effect, as doubling this cell concentration did not increase sensitivity of the assay (i.e. the difference between the basal CPM and peak CPM). Extending the exposure time to tritiated thymidine from 24 to 48 h increased both basal and peak mitogenesis, and also did not present any significant increase in the sensitivity of the assay (figure 4). Therefore, pulsing with tritiated thymidine 24 h prior to cell harvest was deemed to be sufficient. Murray Cod head kidney cells had a broad tolerance to a range of osmolarities (Figure 5). The Murray Cod serum measured by the osmometer had an osmolarity of 289 ± 4 mOsm (n=20), while the optimum range for mitogenic response was between 250-340 mOsm. Culturing cells outside this osmolarity range resulted in a decrease in tritiated thymidine incorporation in both the basal and stimulated cells. At 310 to 340 mOsm, the basal and peak mitogenic responses were less than lymphocytes cultured at 250 to 280 mOsm, however the proliferative index (i.e. peak CPM/ basal CPM) was highest for lymphocytes cultured in an osmolarity of 340 mOsm. Supplementing the TCM with Murray Cod serum at 10% v/v helped support mitogenesis compared to media that did not contain serum (i.e. 1 484 ± 47 versus 214 ± 13 CPM of incorporated tritiated thymidine at 2.5 μg/mL PHA). Heattreated Murray Cod serum supported the highest level of proliferation of Murray Cod head kidney cells (6 416 ± 137 CPM at 2.5 μg/mL PHA), however as both the basal and the PHA-stimulated responses were increased, the proliferative index was not improved. The addition of 2-ME did not aid in the mitogenic response of Murray Cod head kidney cells (figure 6). Peak responses were lower for all groups with the supplement. The removal of HEPES, prior to cell culture, marginally increased the mitogenic response of Murray Cod head kidney cells (Figure 6). The mitogenic response of Murray Cod head kidney cells cultured under the identified optimum conditions (described above), showed that there was variation the responses of individual fish. Nevertheless, the fish used in the optimisation studies were representative of the population and were in the range of the variation (Figure 7). There was no significant difference between the responses of PEG treated fish and the untreated controls, suggesting that PEG is a suitable vehicle for delivering chemicals in lymphoproliferation studies (Figure 7). Phagocytosis Maximum phagocytic activity occurred at 20°C, increasing linearly with incubation time from 15% to a maximum of 25% by 7 d (Figure 8). Granulocytes were generally more robust than lymphocytes and marginal numbers of the cells were lost only at 25°C (Figure 9a). Lymphocytes survived better at cooler temperatures and after 7 d at 15°C, 70% of the lymphocytes remained (Figure 9b). The proportion of debris increased with incubation time and temperature (Figure 9c). 61 AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Immune function assays in Murray Cod V . 12, pp. 57-71, 2006 Vol Harford et al Figure 22. The proliferative response of Murray Cod head kidney cells to 4 mitogens. Cells (2.5x106 mitogens cells/mL) were incubated for 5 d at 15°C. Values represent the mean ± se of triplicate samples from one fish. As it was of interest to enumerate lymphocytes following exposure to chemicals, it was determined that for this assay, the standardised conditions should also support lymphocytes. The conditions for maximum lymphocyte counts and maximum phagocytic activity were observed under 2 experimental conditions, at 2 d and 20ºC as well as at 5 d and 15ºC. Under these conditions lymphocyte counts were approximately 2 500 and phagocytosis was 15%. Although a higher phagocytic activity occurred as time progressed, lymphocytes became less viable in the process. Therefore it was decided that the most effective incubation time and temperature was 2 d at 20oC. There was a linear increase in phagocytosis with increasing cell:bead ratio up to 1:50 when background fluorescence begins to interfere with the detection of beads engulfed by granulocytes (Figure 10). Furthermore, cells incubated at with five different cell:bead ratios, showed no difference in the counts of granulocyte and debris-gated regions, but from 1:25 and 1:50 cells per bead there was a progressive decline in the lymphocyte subpopulation counts (Figure 11). From these results it was determined that a ratio of 1:25 was the best compromise for measuring maximum phagocytosis, while minimising time-induced reductions in lymphocyte counts. Results from the flow cytometry confirmed the findings of the mitogenesis study and suggested that Murray Cod head kidney cells tolerated a broad range of osmolarities i.e. 250 to 340 mOsm (Figure 12a). Culturing of head kidney cells below 250 mOsm resulted in a reduction of lymphocyte counts, while granulocyte counts were lower in the samples cultured at 360 mOsm. The phagocytic activity of Murray Cod head kidney cells was markedly reduced by low osmolarities of 210 62 to 250 mOsm but higher osmolarities of 310 to 340 mOsm increased the number of granulocytes with beads (FITC+ve granulocytes) (Figure 12b). Murray Cod lymphocytes required FCS as a supplement and thus omitting it from the cultures also reduced the phagocytic activity of the head kidney cells (Figure 13). In contrast to the mitogenesis study, lymphocyte counts were lower in all cultures that were supplemented with Murray Cod serum and head-treated Murray Cod serum also reduced phagocytic activity of cell cultures. Although there appeared to be an increase in the phagocytic activity of cultures supplemented with Murray Cod serum that was not heat-treated, culturing cells in this media resulted in the clumping of beads. This resulted in major interference in during flow cytometric analysis, especially in the mean voltage measurements, and was deemed not suitable for the assay. Murray Cod head kidney cells responded well and actively engulfed the fluorescent beads under the identified optimum conditions (described in previously; Figure 14). There were a higher proportion of FITC +ve granulocytes from the untreated fish group compared to the fish used in the optimisation studies. This may be attributed to the lower average granulocyte numbers and the higher average lymphocyte numbers from the untreated fish, however the study suggests that the fish used in the optimisation studies were somewhat representative of the population and that the culture conditions were suitable for measuring phagocytosis in Murray Cod. There was no significant difference between the responses of PEG treated fish and the untreated controls, suggesting that PEG is a suitable vehicle for delivering chemicals in phagocytosis studies (Figure 14). AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Immune function assays in Murray Cod V . 12, pp. 57-71, 2006 Vol Harford et al Figure 3. The response of Murray Cod head kidney cells (2.5x106 cells/mL) to PHA at three temperatures for a) 3 d, b) 5 d, c) 7 d. Values represent the mean ± se of triplicate samples from one fish. 63 AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Immune function assays in Murray Cod V . 12, pp. 57-71, 2006 Vol Harford et al Figure 4. The effect of cell concentration on mitogenic response of Murray Cod head kidney cells with a) 24 h and b) 48 h exposure to tritiated thymidine. Cells were incubated at four different concentrations for 5 d at 15°C. Values represent the mean ± se of triplicate samples from one fish. Figure 55.. Proliferation profiles of Murray Cod head kidney cells incubated at various different osmolarities. Cells (5.0x106 cells/mL) were incubated with PHA for 5 d at 15°C. Values represent the mean±se of triplicate samples from one fish. DISCUSSION Murray Cod were an extremely robust experimental species and there were no deaths due to the transport and handling of the fish and they displayed no signs of disease whilst being held in captivity. Head kidney tissue was targeted for this study because it contains a mixed population of granulocytes (phagocytes) and lymphocytes. Furthermore, the mitogenic response of Murray Cod lymphocytes was strongest in mixed cultures, which is likely to be due to cytokine signalling and support. 64 Mitogenesis This investigation optimised the conditions required for the lymphoproliferation of Murray Cod head kidney cells. However, as previously reported by our group, the native fish lymphocyte responses to mitogenic stimuli were lower compared to other exotic species, such as rainbow trout (O’Halloran et al. 1998) and there was variability between individual fish (O’Halloran 1996). Additionally, the traditional media supplements of 2-ME and HEPES did not enhance the mitogenic responses of Murray Cod lymphocytes. The AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Immune function assays in Murray Cod V . 12, pp. 57-71, 2006 Vol Harford et al Figure 6. The effect of 2-ME on the mitogenic response of Murray Cod head kidney cells. Cells (5.0x106 cells/mL) were incubated with PHA for 5 d at 15°C. Values represent the mean±se of triplicate samples from one fish. which suggests that pooled heat-treated Murray Cod serum could have a detrimental effect on cultured lymphocytes. Figure 7. The effect of HEPES (20 mM) on the mitogenic response of Murray Cod head kidney cells. Cells (5.0x106 cells/mL) were incubated with PHA for 5 d at 15°C. Values represent the mean±se of triplicate samples from one fish. supplementation of media with pooled heat-treated Murray Cod serum increased both the basal and peak proliferation of lymphocytes but did not improve the proliferative index of the assay. Therefore the standardised protocols used 10% heat-treated FCS (providing 3475 ± 92 CPM at 2.5 mg /mL PHA with a proliferation index of 1.5) because FCS batches would be less variable in composition than serum collected and pooled from fish and it was also easier to obtain from a reliable source. Furthermore, under the conditions of the phagocytosis assay, lymphocytes numbers were reduced, Information from flow cytometry suggested that the cellculture media did not fully support the basal growth of lymphocytes, as there was a gradual loss over the incubation period. However, the survival of head kidney lymphocytes from Murray Cod was sufficient at 15°C and gave a consistent, day-to-day, bell-shaped mitogenic dose-response curve when stimulated with PHA. Therefore, PHA-induced mitogenesis was determined to be an appropriate assay for our immunotoxicology studies but it is worth noting that other mitogens may produce a greater response. The mitogen concentrations investigated in this study were the same ranges that stimulated lymphoproliferation in rainbow trout and silver perch, however higher concentrations of LPS and ConA have been used by other researchers (Miller et al. 1986). In addition, phorbol ester/Ca2+ ionophore exposure mimics the phosphotidylinositol bisphosphate signal transduction pathway and would be a candidate for further trials. Such compounds are potent mitogens in other species and do not require macrophage cytokine signalling, which is vital for peripheral blood leukocyte and splenocyte cultures that have very limited macrophage numbers (Lin et al. 1992). Previous studies in our laboratory have shown that RPMI 1640 was the most suitable media for silver perch mitogenesis (O’Hallora 1996), however other media could be investigated in future attempts to further improve the sensitivity of the assay. Consequently, the optimised mitogenesis procedure is described as follows. Standardised protocol for Murray Cod mitogenesis: Incubate Murray Cod head kidney cells (5x106 cells/mL, triplicate wells) for 5 d at 15°C with PHA (10 to 2.5 μg/mL), in 96 well plates, with HEPES-free TCM (200 μL at 280 to 300 mOsm) and an atmosphere of 5% CO2/95% air. Pulse cells by adding 18.5 GBq/well of tritiated thymidine 24 h 65 AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Immune function assays in Murray Cod V . 12, pp. 57-71, 2006 Vol Harford et al Standardised protocol for Murray Cod phagocytosis: Incubate Murray Cod head kidney cells (1x106 cells/mL, in triplicate) with 2.5 x 107 FITC latex beads in TCM with 5% CO2 in air at 20°C for 2 d and then analyse on the flow cytometer. Analyse negative controls (i.e. cells suspension of 1x106 cells/mL incubated with the samples) immediately following the addition of 2.5x107 FITC latex beads. Place counting gates around two subpopulations representing granulocytes and lymphocytes. Also place a gate around debris so that the toxic effects of the test chemicals can be quantitated. Collect a total of 10 000 events per sample and store electronically for future analysis. Figure 8. Effect of incubation temperature on phagocytosis of FITC beads by Murray Cod head kidney cells over a 7 day period at a cell:bead ratio of 1:25. Cells (1x106 cells/mL) were incubated at 15, 20 and 25°C for 2, 3, 5 and 7 d. The values represent the mean ± se of triplicate samples from one fish. prior to harvesting onto glass fibre mats using cell harvester. Quantify tritiated thymidine incorporation into newly divided cells (i.e. proliferation) using a liquid scintillation counter and scintillation fluid. Phagocytosis Flow cytometry was a useful tool, not only in measuring phagocytosis but also for the monitoring of cell subpopulations in the optimisation of culture conditions. Observations made in the mitogenesis studies were confirmed by flow cytometric data, which showed the relative numbers of subpopulations after incubations under various conditions. The only notable exception were the experiments investigating heat-treated Murray Cod serum, where lymphocyte numbers were reduced under the conditions of the phagocytosis assay but proliferation appeared increased in the mitogenesis assay. The flow cytometric analysis of phagocytosis was developed to replace tedious and subjective assessments using light microscopy. The flow cytometry phagocytosis assay correlated well with the method of microscopy counting and was deemed suitable to assay the phagocytic function of head kidney granulocytes (Halford 2004). Moreover, the flow cytometer also has the advantage of offering additional data on the integrity of head kidney subpopulations, which increases the ability of the assay to identify immunotoxicity. Therefore the optimised phagocytosis procedure is described as follows. 66 These studies describe the first experiments investigating the immune function of Murray Cod. The assays standardised in this paper were used to culture the head kidney cells from a number of untreated and PEG treated Murray Cod. The results show that the optimised culture conditions are suitable for measuring phagocytosis and mitogenesis, and that the fish used in the optimisation studies were representative of the population (Figures 7 and 14). However, variation in the mitogenic responses of individual Murray Cod suggest that this parameter should be standardised using the proliferation index, i.e. peak CPM/basal CPM. These assays will be useful to aquaculturalists and ecotoxicologists interested in monitoring the immune responses of Murray Cod following exposure to xenobiotics. The Murray Cod aquaculture industry is growing and fish will become easier to obtain in the future. Additional industry partners will emerge as they become more interested in the physiological systems of the fish that they are farming and see the need for specific biological tools to be developed to monitor the health of stock animals. Murray Cod are also an excellent species for ecotoxicological testing because they are the top predator of the Murray-Darling basin, they adapt extremely well to laboratory conditions and provide an excess of tissue to work with. AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Immune function assays in Murray Cod V . 12, pp. 57-71, 2006 Vol Harford et al Figure 9. The effect of incubation time and temperature on subpopulation and debris counts. a) Granulocytes, b) Lymphocytes, c) Debris. Cells (1x106 cells/mL) were incubated at 15, 20 and 25°C for 2, 3, 5 and 7 d. The values represent the mean ± se of triplicate samples from one fish. 67 AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY V . 12, pp. 57-71, 2006 Vol Immune function assays in Murray Cod Figure 10. The effect of cell:bead ratio on the phagocytosis of FITC latex beads by Murray Cod head kidney cells. Cells (1x106 cells/mL) were incubated at 15°C for 2, 3, 5 and 7 d. The values represent the mean ± se of triplicate samples from one fish. Figure 11. The effect of cell:bead ratio on subpopulation counts of Murray Cod head kidney cells. a) Granulocytesand b) Lymphocytes and debris. Cells (1x106 cells/mL) were incubated for 3 d at 20°C. Values represent the mean ± se of triplicate samples from one fish. 68 Harford et al AUSTRALASIAN JOURNAL OF ECOTOXICOLOGY Immune function assays in Murray Cod V . 12, pp. 57-71, 2006 Vol Harford et al Figure 13. The effect of serum supplements on a) the subpopulation counts and b) phagocytic activity of Murray Cod head kidney cells. Data is represented as percentage of control samples (RPMI with 10% FCS) i.e. granulocytes 6300 ± 250 counts, lymphocytes 2300 ± 150counts, FITC+ve granulocytes 10 ± 0.5% and mean voltage 126 ± 9 volts. 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