Bull Vet Inst Pulawy 53, 241-246, 2009
EFFECT OF β-1,3/1,6-D-GLUCAN
ON THE PHAGOCYTIC ACTIVITY
AND OXIDATIVE METABOLISM
OF PERIPHERAL BLOOD GRANULOCYTES
AND MONOCYTES IN RATS
ROMAN WÓJCIK, EWA JANOWSKA,
JOANNA MAŁACZEWSKA, AND ANDRZEJ KRZYSZTOF SIWICKI
Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine,
University of Warmia and Mazury in Olsztyn, 10-957 Olsztyn, Poland
[email protected]
Received for publication December 12, 2008
Abstract
The experiment was performed on 20 adult Wistar rats aged 12 weeks, divided into two equal groups (control and
experimental), each comprised of five males and five females. From the first day of the experiment, the experimental group rats were
fed Murigran feed supplemented with β-1,3/1,6-D-glucan at a dosage of 12-19 mg/rat/d, subject to body weight, while the controlgroup was administered the same feed without any additives. At the beginning of the experiment and then after 14 days, arterial
blood samples were collected from the rats and diluted with heparin to measure and compare the phagocytic activity and oxidative
metabolism of peripheral blood granulocytes and monocytes by flow cytometry. Statistically higher levels of the activity were
observed in the group of rats administered glucan than in controls, expressed in terms of the percentage of phagocytic cells as well as
average fluorescence intensity. β-1,3/1,6-D-glucan also had a positive effect on the oxidative metabolism of both granulocytes and
monocytes after stimulation with E. coli, and on the oxidative metabolism of granulocytes after stimulation with PMA.
Key words: rats, granulocytes, monocytes, β-1,3/1,6-D-glucan, phagocytic activity, oxidative metabolism.
Immunomodulation is a process, which
stimulates or suppresses the immune system's response
to the administered drugs or chemical compounds (14).
The above applies to all pharmacological products and
xenobiotics affecting the immune system. Subject to the
induced effect, immunomodulators are divided into
immunostimulators, which augment or restore the
immune response, and immunosuppressants that
suppress or inhibit the immune response (10, 22, 28). In
view of their biological origin, immunomodulators are
further divided into standard animal proteins and
isolated substances purified of microbes and post-culture
bacterial liquid (33).
Synthetic and natural immunomodulators are
widely applied in the prevention and treatment of
infections, non-infectious diseases, and neoplastic
diseases, while some immunomodulators that suppress
immune
system
reactivity
(pharmacological
immunosuppression) are used in transplantology, thus
furthering the development of this medicinal science (9,
28). These types of drugs are also used in the treatment
of autoimmunological diseases.
The significance of immunostimulation is
growing, due to the immunosuppressive impact of
environmental pollution and to the suppressive effect of
popular drugs on immunity factors in animals (9).
Immunostimulators have a stimulating effect on nonspecific cellular and humoral immune mechanisms and a
specific immune response. There is a very broad range
of known and applied immunostimulators, but the search
for new ones continues. Researchers aim to obtain
compounds with a stronger effect that eliminate or
significantly minimise the resulting side effects. The
intensity of the desired effect is largely dependent on the
length of the immunomodulator's retention in the body.
For this reason, scientists are searching for derivatives of
known modulators, which are not quickly eliminated
(17).
The objective of this study was to determine the
effect of β-1,3/1,6-D-glucan on the phagocytic activity
and oxidative metabolism of peripheral blood
granulocytes and monocytes in rats.
242
Material and Methods
The experiments were carried out in
conformance with the Animal Protection Law and the
recommendations of the Animal Ethics Committee of
the University of Warmia and Mazury in Olsztyn.
During the experiments, animals were kept on Faculty
premises with the observance of adequate experimental
conditions.
Experimental design. The experimental
material comprised 20 adult Wistar rats aged 12 weeks,
divided into two equal groups (control and
experimental), consisting of five males and five females
each. Over a period of two weeks starting on the first
day of the experiment, the experimental group rats were
fed Murigran feed supplemented with β-1,3/1,6-Dglucan (Biolex Beta-HP) at a dosage of 12-19 mg/rat/d,
subject to body weight, while the control group animals
were administered the same feed without any additives.
At the beginning of the experiment and then after 14
days, arterial blood samples were collected from rats and
diluted with heparin.
Measurement of phagocytic activity. The
phagocytic activity of granulocytes and monocytes was
determined with the use of Phagotest (Orpegen Pharma).
All test reagents were prepared in accordance with the
manufacturer's
recommendations.
One
hundred
microlitres of whole blood chilled to 0ºC and 20 µl of
chilled bacteria were added to each of the two 5 ml test
tubes (control and experimental) and shaken for around
3 s at low speed. The experimental sample was
incubated for 10 min at 37ºC, and the control sample in
an ice bath at 0ºC. After incubation, 100 µl of quenching
solution was added to each sample and the samples were
shaken. Three millilitres of washing solution chilled to
0ºC were added, the samples were centrifuged for 5 min
at 4ºC (250 x g), and the supernatant was removed. The
rinsing procedure was performed twice, and 2 ml of
lysing solution at room temperature were added to each
sample. The samples were shaken and incubated at room
temperature for 20 min. The samples were then
centrifuged for 5 min at 4ºC (250 x g) and the
supernatant was removed. Three millilitres of washing
solution chilled to 0ºC were added to each sample, the
samples were centrifuged for 5 min at 4ºC (250 x g), and
the supernatant was removed. Two hundred microlitres
of DNA staining solution chilled to 0ºC was added and
the samples were shaken and incubated for 10 min in an
ice bath. Cellular phagocytic activity was measured in a
cytometer (Beckmnan Coulter, Epics XL) less than 60
min after the last reagent had been added. The Phagotest
was performed with the involvement of fluoresceinstained E. coli strains, which were phagocytised by
macrophages. Cell nuclei were also stained. The test
determines the number of phagocytising granulocytes
and monocytes separately, and their phagocytic activity,
i.e. the number of bacteria absorbed by a single cell in
terms of fluorescence intensity.
Measurement of oxidative metabolism. The
oxidative metabolism of granulocytes and monocytes
was performed by the use of Bursttest (Phagoburst,
Orpegen Pharma). All test reagents were prepared in
accordance with the manufacturer's recommendations.
Each analysed sample of whole heparinised blood was
divided into four test tubes of 100 µl each and chilled to
0ºC. Twenty microlitres of chilled E. coli strains were
added to the first sample (experimental), 20 µl of
washing solution was added to the second sample
(negative control), 20 µl of N-formyl-met-leu-phe
(fMLP) was added to the third sample (low control), and
20 µl of PMA was added to the fourth sample (high
control). All test tubes were mixed and incubated for 10
min at 37ºC (excluding the fMLP sample, which was
incubated for 7 min). After the incubation, each test tube
was supplemented with 20 µl of substrate solution and
was thoroughly shaken. All samples were incubated for
10 min at 37ºC. After the incubation, 2 ml of lysing
solution at room temperature was added. Test tubes were
shaken and incubated at room temperature for 20 min.
All samples were centrifuged for 5 min at 4ºC (250 x g),
and the supernatant was removed. All test tubes were
rinsed once with 3 ml of washing solution, centrifuged
for 5 min at 4ºC (250 x g), and then the supernatant was
removed. Two hundred microlitres of staining solution
chilled to 0ºC was added to each sample, and test tubes
were shaken and incubated for 10 min in an ice bath.
Intracellular killing activity of phagocytes was
determined in a cytometer (Beckmnan Coulter, Epics
XL) in less than 30 min after the last reagent had been
added. Three activators were used for cell stimulation:
E. coli strains, PMA (4-phorbol-12-β-myristate-13acetate) as the strong activator, and fMLP as the weak
activator. The added dihydrorodamine (123-DHR) was
oxidised in mitochondria by H2O2 resulting from cell
stimulation and was converted to cation rhodamine 123
(R123), the fluorescent emitter.
The obtained results were processed
statistically in a one-factorial analysis of variance in an
orthogonal design. The significance of differences
between groups was verified by the Student's t-test with
the use of GraphPad Prism 5 software.
Results
The analysis of the obtained results has shown
that the phagocytic activity of granulocytes was
significantly higher (P≤0.05) in the group of rats
administered β-1,3/1,6-D-glucan than in control, both in
terms of the percentage of phagocytising cells and
average fluorescence intensity. The percentage of
phagocytic monocytes and their phagocytic activity,
expressed in terms of average fluorescence intensity,
was also statistically higher in the group of rats
stimulated with the glucan than in controls (Table 1).
β-1,3/1,6-D-glucan also had a positive effect on
the oxidative metabolism of granulocytes after
stimulation with E. coli, where the percentage of
stimulated cells was significantly higher in this group
than in the control, while average fluorescence intensity,
although higher in the group of rats stimulated with β1,3/1,6-D-glucan than in control, did not produce
statistically significant differences (Table 2).
243
Table 1
Percentage of phagocytic granulocytes and monocytes and average fluorescence intensity of granulocytes
and monocytes in each rat group measured in the Phagotest
Granulocytes
Monocytes
Average
Average
Phagocytic cells (%)
fluorescence
Phagocytic cells (%)
fluorescence
intensity
intensity
Control
31.22 ±2.15
58.27 ±1.43
24.18 ±1.34
82.22 ±0.54
β-glucan
46.55 ±1.55***
67.17 ±1.03**
35.04 ±0.94***
93.64 ±0.95***
** - P≤0.01; *** - P≤0.001
average
fluorescence
intensity
% stimulated
cells
average
fluorescence
intensity
% stimulated
cells
average
fluorescence
intensity
Control
β-glucan
% stimulated
cells
Table 2
Average intracellular killing activity of granulocytes and average fluorescence intensity in each rat group after
stimulation with fMLP, PMA, and E. coli as determined in the Bursttest
Granulocytes
fMLP
PMA
E. coli
21.51 ±1.26
19.57 ±0.84
1.53 ±0.44
2.04 ±0.40
83.32 ±1.90
90.71**±1.10
7.41 ±0.51
8.36 ±0.60
82.58 ±2.80
89.99*±0.46
13.22 ±0.43
11.92 ±0.44
∗ - P<0.05; ∗∗ - P<0.01
7.83 ±1.10
9.12 ±0.52
8.84 ±0.80
9.70 ±1.10
average
fluorescence
intensity
45.78 ±1.24
47.56 ±1.40
% stimulated
cells
average
fluorescence
intensity
8.6 ±1.20
9.23 ±0.90
% stimulated
cells
Control
β-glucan
average
fluorescence
intensity
% stimulated
cells
Table 3
Average intracellular killing activity of monocytes and average fluorescence intensity in each rat group after stimulation
with fMLP, PMA, and E. coli as determined in the Bursttest
Monocytes
fMLP
PMA
E. coli
42.57 ±1.30
7.98 ±0.72
***
51.79 ±0.72 10.75** ±0.40
∗∗ - P<0.01; ∗∗∗ - P<0.001
Following monocyte stimulation with E. coli,
oxidative metabolism, expressed as the percentage of
stimulated cells and average fluorescence intensity, was
significantly higher in the group of rats stimulated with
β-glucan than in control (Table 3).
A similar effect to that noted after granulocyte
and monocyte stimulation with E. coli was observed
after PMA stimulation (strong respiratory-burst
activator). Although the level of oxidative metabolism
of granulocytes and monocytes was higher in the group
of rats administered β-1,3/1,6-D-glucan than in the
control (Tables 2 and 3), in both cases a statisticallysignificant increase was reported only in reference to the
percentage of stimulated granulocytes in the group of
animals receiving β-glucans (Table 2).
Although the investigated parameters were
higher in the group stimulated with β-1,3/1,6-D-glucan
than in the control group after granulocyte and
monocyte stimulation with fMLP (weaker respiratoryburst activator), statistically significant differences were
not observed in the percentage of stimulated
granulocytes and monocytes or the intensity of those
cells' oxidative metabolism (Tables 2 and 3).
Discussion
The preventive and therapeutic effectiveness of
drugs modulating the immune system of various animal
species has been investigated in numerous laboratory
studies and clinical trials for many years. A special
244
attention should be paid to biostimulants (18, 28),
immunostimulants of natural origin, which may be
effectively used in organic animal farms where the
animals' diets are not supplemented with synthetic
stimulants. Organic farming shows a preference for noninvasive treatment methods, including the administration
of immunity stimulators, wherever possible, in the most
convenient form of feed supplements. One of the most
popular feed supplements are β-glucans (23, 28, 29, 38),
including β-1,3/1,6-D-glucan, which is found in the cell
wall of Saccharomyces cerevisiae yeast species. The
branched β-1,3/1,6-glucan polysaccharide found in the
Biolex-Beta HP product has the structure of a β-1,3
main chain connected to a β-1,6 side chain. This
polysaccharide is isolated from mannoproteins without
the use of aggressive alkalines or acids in the hydrolysis
process, and it occurs in an unmodified, natural form
that guarantees the highest level of biological activity
(36). Research results have shown that β-glucans
significantly stimulate the immunity to viral, bacterial,
fungal, and parasitic infections of many animal species
(2, 16, 27, 35, 37). β-1,3/1,6-glucan's ability to activate
the immune system was investigated in detail. It was
found to offer effective protection of animals against
Gram-positive and Gram-negative bacteria, viruses,
fungi, and parasites in numerous experiments (11).
Initially, β-glucan was believed to be a strong stimulator
of the interplasmic system, and its ability to activate
non-specific defence mechanisms in macrophages,
phagocytising cells, was discovered only later (21).
In this experiment, a significant increase in the
percentage of phagocytic cells and average fluorescence
intensity of monocytes and granulocytes (Phagotest) was
observed in comparison with the control in the
experimental group of rats fed a diet supplemented with
β-1,3/1,6-D-glucan (Table 1). Similar results were
reported by other authors in studies of mice (24),
shrimps (4), and lambs (36), and they noted a 30%-50%
increase in intracellular killing activity of blood
phagocytes in the group of animals fed diets
supplemented with β-1,3/1,6-D-glucan. In in vitro and in
vivo experiments, Estrada et al. (7) have demonstrated
that β-glucan isolated from oats had an
immunostimulatory effect on immunocompetent cells in
mice, activating, among others, mouse macrophages and
boosting the animals' resistance to diseases caused by
selected pathogens, including Staphylococcus aureus
and Eimeria vermiformis. According to the results of the
experiment conducted on mice by SzymańskaCzerwińska et al. (31) the highest level of macrophage
phagocytic activity was observed after oral as well as
intravenous administration of β-glucans. The exact
mechanism of β-glucan's effect on the functioning of
phagocytic cells has not yet been fully explained, but
researchers have demonstrated that by binding to, for
example, CR3 receptors or TLR (Toll-like receptors), βglucans activate phagocytes.
In this study, β-glucan also had a positive effect
on the intracellular killing activity of granulocytes and
monocytes stimulated with E. coli as well as
granulocytes stimulated with PMA, expressed as a
higher percentage of stimulated cells and higher average
fluorescence intensity (Tables 2-3). An increase in the
above parameters is indicative of higher activity of
phagocytes against bacteria and more effective
elimination of pathogens. Similar results were reported
by Wójcik et al. (36) in a study of sheep, where the
respiratory-burst activity (RBA) of phagocytes increased
significantly in the experimental group receiving βglucan in comparison with the control. Research
findings reported by Suzuki et al. (30) coincide with the
results of this study due to increased production of
hydrogen peroxide (H2O2) and increased activity of
lysosomal enzymes in mouse macrophages after oral
administration of β-glucan. In a study conducted by
Brattgjerd et al. (1) macrophages isolated from the renal
cortex and stimulated with both β-glucan and PMA
increased hydrogen peroxide levels. As noted by Ohno
et al. (20) and Tikunaka et al. (33) β-glucan also
stimulated the production of nitric oxide (NO), another
RBA factor, and activated lysosomal enzymes (29).
Similar in vivo experiments carried out by Brattgjerd et
al. (1) have shown that β-glucan induces and increases
the phagocytic ability of macrophages to kill bacteria by
raising H2O2 levels in the Atlantic salmon after
stimulation with Vibrio salmonicida.
β-glucan has a varied effect on the activity of
phagocytic cells, subject to the manner of
administration, dosage and the period of treatment, as
observed by Sakurai et al. (24) in an experiment
conducted on mice. When β-glucan was supplied
intravenously, macrophages increased the production of
H2O2 and interleukin 1 (IL-1). The above was not
reported in the group of mice injected with β-glucan
intraperitoneally. According to Suzuki et al. (30) oral
administration of β-glucan induced the production of
H2O2 and IL-1 in mouse macrophages, stimulating their
phagocytic activity in comparison with control. Other
studies have confirmed that the above methods of βglucan administration are also capable of stimulating
respiratory-burst activity of phagocytes in fish.
Intraperitoneal administration increased phagocyte RBA
in different fish species, including the Atlantic salmon
(Salmo salar) (1, 5), the rainbow trout (Oncorhynchus
mykiss) (13), and the turbot (Psetta maxima) (25). When
applied orally, yeast β-glucans increased the respiratoryburst activity of leukocytes in the rainbow trout (26),
turbot (34), and North African catfish (Clarias
gariepinus) (39). The results of in vivo experiments have
shown that β-glucans stimulate the production of
superoxide anions by macrophages in the Atlantic
salmon (6, 12), the rainbow trout (19), and the turbot
(8).
Incubation time also contributes significantly to
an increase in the respiratory-burst activity of
phagocytes. As noted by Jorgensen et al. (12) the
presence of β-glucans in the incubation medium
enhances respiratory-burst activity and proliferation of
macrophages in the Atlantic salmon only after 7 d.
Castro et al. (3) observed that the respiratory-burst
activity of cells incubated for 1, 3 and 6 h, with no PMA
stimulation, increased significantly with a simultaneous
rise in β-glucan levels. However, after stimulation with
PMA, cells incubated at high β-glucan concentrations
245
were marked by similar or even lower levels of
respiratory-burst activity in comparison with the control,
which was not treated with β-glucan. The response to
PMA stimulation could be related to the place of its
activity, i.e. the preserved or modified phosphorylation
capacity of NADPH oxidase cytosolic factor (15). Tahir
et al. (32) discovered that β-glucan doses lower than 1
mg/ml optimise the level of respiratory-burst activity of
macrophages (Limanda limanda, L.) already after 24-48
h of incubation.
The results of this study pave the way for
follow-up research into practical applications of βglucans in the immunoprophylaxis in animals and
humans, particularly in the restoration of impaired
immunity in periods of increased incidence of bacterial
and viral infections.
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