Download Characterization of Blood Leukocytes in Fish Species of

Characterization of Blood Leukocytes in Fish Species of Special Concern by Rapid
Cytochemical Staining
Dušan Palić1, Jelena Palić2 , Linda S. Beck3
Department of 1 Biomedical Sciences, 2Veterinary Pathology, The College of Veterinary Medicine ,Iowa State University; 3US Fish & Wildlife Service –Malherur Wildlife Refuge
Abstract
Materials and methods
Studies of innate immunity in fish species of special concern are essential for
better understanding of their health status. The cytochemical and
morphological characterization of blood leukocytes has been used to provide
information about phylogenetic differences and determine potential for use of
neutrophil functional assays. Rapid, simple, cytochemical staining kits used
routinely for staining mammalian leukocytes have been used to characterize
leukocytes from blood of four fish species: Arctic grayling, cutthroat trout,
June sucker, and shovelnose sturgeon. Three fish from each species were
sampled and six blood smears were prepared from each fish. Blood smears
were stained with Peroxidase 391 (myeloperoxidase, MPO), Sudan Black B
(SBB), Periodic acid-Schiff (PAS), alkaline phosphatase (AP), alpha-naphthyl
acetate esterase, and Diff-quick stain; examined using bright field and
differential interference contrast microscopy, and leukocytes on blood smears
evaluated based on the cell morphology
morphology, and presence or absence of the
specific chromogen. Presence of lymphocytes, monocytes,
platelets/thrombocytes and granulocytes was determined in all fish species.
Arctic grayling, June sucker, and cutthroat trout had MPO positive
granulocytes, while shovelnose sturgeon did not. Sturgeon granulocytes
showed positive reaction for leukocyte AP. Presence of MPO indicated
potential to measure oxidative burst and degranulation of neutrophil primary
granules in Arctic grayling, cutthroat trout and June sucker. Absence of MPO
in shovelnose sturgeon suggested use of different enzyme marker (AP) in
degranulation assay, as well as use of phagocytosis or bacterial killing assays
as method of choice for this species. Standardized cytochemical techniques
allowed for rapid screening of leukocytes, reducing the number of fish, time
and effort to select adequate neutrophil function assays to be used in studies
of health status in species of special concern.
Leukocytes from four fish species of special concern were evaluated: Arctic
grayling (Thymallus arcticus), cutthroat trout (Oncorhynchus clarki lewisi),
June sucker (Chasmistes liorus), and shovelnose sturgeon (Scaphirhynchus
platorynchus). Shovelnose sturgeon were used as surrogates for the
endangered pallid sturgeon (S. albus). Three fish from each species were
euthanized in MS-222, blood was collected in heparinized microhematocrit
tubes from severed caudal peduncle, and six blood smears per fish were
prepared. Blood smears were stained with Peroxidase 391
(myeloperoxidase, MPO), Sudan Black B (SBB), Periodic acid-Schiff (PAS),
alkaline phosphatase (AP), alpha-naphthyl acetate esterase (α-NAE) (all
from Sigma), and Diff-quick (Fisher) according to manufacturer’s
instructions, with minor modifications. Leukocytes were examined using
bright field and differential interference contrast microscopy
microscopy. Cell morphology
and presence or absence of the specific chromogen were evaluated.
For granulocyte function assays kidney tissue from four individual cutthroat
trout and shovelnose sturgeon was aseptically collected, and granulocytes
were separated using a previously described technique [1]. Degranulation
(measured by detection of MPO) and oxidative burst (measured by
reduction of cytochrome C) were determined as previously described [1, 2].
The release of AP from shovelnose sturgeon granulocytes was determined
using AP detection kit (Biosciences) in a setup similar to [1]. The percent
release of MPO and AP was calculated using following formula:
% release = [(ODstimulated - ODbackground) / (ODlysed – ODbackground)] x 100
*
Figure 1. Characteristic morphology of blood neutrophils. Cells with multi-lobed or indented
nucleus with moderate nuclear to cytoplasmic ratio were determined by Diff quick staining in
Arctic grayling (A), cutthroat trout (B), and June sucker (C). Presence of myeloperoxidase in
neutrophils from Arctic grayling (D), cutthroat trout (E), and June sucker (F) was determined
using Peroxidase 391 staining kit. Bar = 20 µM.
*
Figure 3. Degranulation detected as release of myeloperoxidase in cutthroat trout (blue)
and shovelnose sturgeon (red) head kidney leukocyte cell suspension after stimulation for
40 min with 1 µg mL-1 Lipopolysaccharide from E. coli (LPS); 1 µg mL-1 Phorbol myristate
acetate (PMA); 5 µg mL-1 Calcimycin A23187; 200 µg mL-1 purified β-glucan from baker’s
yeast; with (+) and without 2.5 µg mL-1 of Cytochalasin B; Hank’s Balanced Salt Solution
with Calcium and Magnesium (HBSS) was used as non-stimulated control, and cells (100%
enzyme activity) were lysed with 0.02% CTAB. *Significant differences (P<0.01) between
stimulated and non-stimulated cells.
Results
*
*
Introduction
Neutrophils are an important component of host defense against many
bacterial, viral and fungal infections, and the evaluation of neutrophil function
is valuable tool for assessment of the health status of individuals and animal
populations. Severe or chronic stress is often associated with poor
performance and has long been associated with immunosuppression in
cultured fish. The decline in population and ecological relevance have placed
fish species of special concern under federal or state conservation programs,
including restocking of the hatchery spawned fish to natural habitats. Artificial
reproduction
d ti off the
th species
i off special
i l concern and
d culturing
lt i ffry tto th
the
stocking size in hatchery environment has been performed with limited
success. Optimal water quality, nutrition, and spawning conditions for these
species are poorly known, and inadequate environment may lead to stress
that could undermine efforts for successful re-introduction of adults and
fingerlings to their native habitats, and weaken immune system of the brood
stock. Cytochemical characterization of leukocytes, and especially
granulocytes, with rapid staining kits detecting different enzymes has been
used in mammalian species to provide insight in leukocyte function. Presence
or absence of granular enzymes can reveal differences in function and type of
the leukocyte, and help in making the decision about which assays show
more promise in evaluation of specific cell type function. Narrowing the choice
potential function assays
y can reduce the number of fish used in the assay
y
of p
optimization.
*
Figure 2. Characteristic morphology and cytochemical staining of the
shovelnose sturgeon blood granulocytes. Presence of eosinophils and
heterophils was detected in blood using Diff quick stain (A). Absence of MPO
(B) and presence of AP (C) in granulocytes indicated the potential for use of AP
in the degranulation assay. Presence of abundant granules in sturgeon
leukocytes was detected by PAS staining (D). Bar = 20 µM.
Figure 4. Oxidative burst detected as Cytochrome C reduction in cutthroat trout (blue) and
shovelnose sturgeon (red) head kidney leukocyte cell suspension after stimulation for 40
min with 1 µg mL-1 Lipopolysaccharide from E. coli (LPS); 1 µg mL-1 Phorbol myristate
acetate (PMA); 5 µg mL-1 Calcimycin A23187; 200 µg mL-1 purified β-glucan from baker’s
yeast; with (+) and without 2.5 µg mL-1 of Cytochalasin B; Hank’s Balanced Salt Solution
with Calcium and Magnesium (HBSS) was used as non-stimulated control. *Significant
differences (P<0.01) between stimulated and non-stimulated cells.
Conclusion
Statement of problem
The effects of captivity and spawning stress on innate immunity in species of
special concern has not been studied, and basic characterization of
granulocytes in these species has not been performed. The limited population
and high value of individual specimens poses a limitation to the sample size
to be used in optimization and determination of the appropriate function
assays in evaluation of the innate immune status of the fish. Cytochemical
staining of granulocytes was performed with commercially available leukocyte
staining kits to determine presence or absence of enzymes characteristic for
granulocytes in other species. The differences in granular enzymes indicated
potential differences in function of the examined cells, assisted in selection of
the assays, and reduced number of fish used to determine appropriate
function assays.
The microscopic analysis of blood smears revealed presence of several
leukocyte cell types in all examined fish species: lymphocytes, monocytes,
thrombocytes, and granulocytes (Tab. 1). Granulocytes in Arctic grayling,
cutthroat trout and June sucker have characteristic neutrophil morphology,
kidney shaped, indented, or multi-lobed nucleus with a moderate nuclear to
cytoplasmic ratio (Fig. 1A-C). The presence of MPO in the cytoplasmic
granules confirmed the presence of neutrophilic granulocytes in Arctic
grayling, cutthroat trout and June sucker blood (Fig. 1D-F), while
eosinophilic granulocytes, and multi-lobed, neutral stained granulocytes
were detected in sholvelnose sturgeon (Fig. 2A). The absence of eosinophil
peroxidase and MPO in sturgeon blood and head kidney granulocytes was
indicative of heterophils
heterophils, rather then neutrophils (Tab
(Tab. 1
1, Fig
Fig. 2B)
2B).
Presence of MPO in neutrophil granules of Arctic grayling, cutthroat trout
and June sucker indicated functional capabilities for degranulation and
oxidative burst, that was confirmed when cutthroat trout head kidney cells
were assayed (Figs. 3, 4). Absence of MPO in shovelnose sturgeon
suggested reduced capabilities for oxidative burst and indicated the need for
a different enzymatic marker to be used in the degranulation assay (Figs. 3,
4). The presence of AP and granules was detected (Fig. 2C-D), and AP as a
marker in the degranulation assay resulted in the detection of 55%
degranulation after 30 min stimulation (Fig. 5).
Table 1. Overview of different leukocytes found in blood of four fish species. All species had
thrombocytes, monocytes/macrophages, and lymphocytes present in the blood. Myeloperoxidase
positive granulocytes were considered neutrophils, and MPO negative granulocytes were considered
heterophils. Eosinophils were detected by Diff quick in shovelnose sturgeon, and no basophils were
detected in any of the fish. The small number of samples and low numbers of blood eosinophils and
basophils could have prevented detecting those granulocyte types in other fish species.
Figure 5. Degranulation detected as activity of alkaline phosphatase in the
supernatant of shovelnose sturgeon head kidney leukocyte cell suspension
stimulated with 5 µg mL-1 Calcimycin A23187; HBSS with Calcium and
Magnesium
g
was used as non-stimulated control. Enzyme
y
activityy in
supernatants of cells lysed with 0.02% CTAB was used as 100%.
Rapid cytochemical staining can be used as an efficient method for
differentiation of various leukocyte types in fish. Information about presence
or absence of the myeloperoxidase is indicative of the capability of the
granulocytes to develop an oxidative burst response to stimulation, while
presence of granules and detection of granular enzymes indicates the most
appropriate marker to be used in the degranulation assays. Therefore,
cytochemical staining performed on limited number of individual fish can
reduce the overall number of fish used to select appropriate granulocyte
function assays for measuring effects of stress on species of special
concern held in captivity.
References
1.
2.
Palić D, Andreasen CB, Menzel BW, and Roth JA (2005). A rapid, direct assay to measure
degranulation of primary granules in neutrophils from kidney of fathead minnow (Pimephales
promelas Rafinesque, 1820). Fish and Shellfish Immunology 19 (3), 217-227.
217 227.
Palić D, Andreasen CB, Herolt DM, Menzel BW, and Roth JA (2006). Immunomodulatory
effects of β-glucan on neutrophil function in fathead minnows (Pimephales promelas
Rafinesque, 1820). Developmental and Comparative Immunology 30(9), 817-830.
This work was sponsored in part by
Western Area Power Administration
grant and Summer Research Scholar
Program, The College of Veterinary
Medicine, Iowa State University
www.sf.adfg.state.ak.us
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