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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
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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
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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
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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
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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.
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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
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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
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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).
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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
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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).
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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.
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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.
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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
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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
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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.
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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
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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.
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Harford et al
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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. Values present the mean ±
se of triplicate samples from one fish.
Figure 14. The phagocytosis and cell counts of Murray cod head kidney cells. Cells were incubated under the optimised
conditions described in 4.2. The values represent the mean ± se (n = 8).
69
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Dean JH and Murray MJ. 2001. Chapter 9: Toxic responses
of the immune system. In Casarett & Doull’s Toxicology: The
Basic Science of Poisons
Poisons. Doull J, Klaassen CD and Amdur
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DeKoning J and Kaattari S. 1991. Mitogenesis of rainbow trout
peripheral blood lymphocytes requires homologous plasma
for optimal responsiveness.
responsiveness In Vitro Cell Developmental
Biology 27A, 381-386.
DEH (Department of Environment and Heritage). 2005.
Murray Cod ((Maccullochella peelii peelii). [Australian
Government website]. http://www.deh.gov.au/biodiversity/
threatened/publications/murray-cod.html#download. Last
Accessed 23/5/2005.
Faulmann E, Cuchens MA, Lobb CJ, Miller NW and Clem
LW. 1983. An effective culture system for studying in
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Transactions of American Fisheries Society 112, 673-679.
Harford AJ, O’Halloran K and Wright PFA. 2006. Flow
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phagocytosis in three Australian freshwater fish
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Figure 12. The effect of osmolarity on a) the subpopulation counts
and b) phagocytic activity of Murray Cod head kidney cells. Data
is represented as percentage of control samples (290 mOsm) i.e.
granulocytes 6700 ± 60 counts, lymphocytes 2100 ± 60 counts,
FITC+ve granulocytes 16 ± 1% and mean voltage 191 ± 34 volts.
Values present the mean±se of triplicate samples from one fish.
ACKNOWLEDGEMENTS
This research was funded by an Australian Research Council
(ARC) Large Grant (No.19803567) awarded to P.F.A.
Wright.
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