Immunology and Cell Biology (1999) 77, 395–403

Research Article
The effect of molecular weight and β-1,6-linkages on priming of
macrophage function in mice by (1,3)-β-D -glucan
JA N E L L E A C L E A RY, 1 * G R A H A M E K E L LY 2 a n d A L A N J H U S BA N D 1
1
Department of Veterinary Anatomy and Pathology, University of Sydney and 2Novogen Ltd, North Ryde,
New South Wales, Australia

Summary 1,3-β-D-glucans (glucans) are structural elements in the cell walls of yeast and fungi with
immunomodulatory properties, mediated through their ability to activate macrophages. This study assessed the acti-
vation of cells of the peritoneal cavity between 3 and 90 days after i.p. injection of particulate yeast glucan differ-
ing in molecular weight (MW) and degree of (1,6)-linkages. Female QS mice, 7–9 weeks of age, were injected, i.p.,
with varying doses of low (< 5 × 105), medium (1–2 × 106) or high (> 3 × 106) MW glucans, all with low (< 5%)
β-(1,6)-linkages, or high MW (> 3 × 106) glucan with high 1,6-linkages (> 20%). All glucans induced a transient
increase in the proportion of neutrophils and eosinophils and a reduction in mast cell numbers in the peritoneal
cavity. Peritoneal macrophages showed an altered morphology, increased intracellular acid phosphatase, increased
LPS-stimulated NO production and increased PMA-stimulated superoxide production. There were no significant
changes in serum lysozyme levels. Most macrophage activities returned to control levels by 28 days post injection
of 1,3-β-D-glucan. There was a trend for higher MW or (1,6)-linked, (1,3)-β-D-glucans to be more stimulatory. It
was concluded that particulate yeast (1,3)-β-D-glucan is an effective stimulator of immune function, the efficiency
of which may be influenced by the MW and degree of (1,6)-linkages.

Key words: glucan, immunomodulation, macrophage activation, particulate, structure.

Introduction The extent to which alterations in the chemical structure

(1,3)-β-D-Glucans (glucans) are a heterogeneous group of
glucose polymers present as structural elements in the cell
walls of yeast, fungi and cereal plants. Several reports over the
past 30 years have described their role as biological response
modifiers and potential immunotherapeutics. Biological activ-
ities associated with in vivo use of these molecules include
tumour inhibition,1 enhanced defence against bacterial chal-
lenge,2,3 increased haemopoeitic activity and radioprotective
effects4 and improved wound healing.5 In vitro, these mole-
cules have been reported to influence macrophage morphol-
ogy,6 release of cytokines (such as TNF-α, IL-6 and IL-1),7
release of nitric oxide,8 lysosomal enzyme secretion,9 hydro-
gen peroxide release,10 arachidonic acid metabolism11 and
alternate pathway complement activation.12
The major mechanism by which glucans are thought to
elicit these responses is through activation of macrophages,
possibly via a macrophage specific (1,3)-β-D-glucan recep-
tor,13 or through binding to the complement receptor CR3
(CD11b/CD18).14 However, recent reports have demonstrated
that glucan can also directly influence the activity of other
immune cells, including T and B cells,15 NK cells,16
eosinophils17 and neutrophils.18
Correspondence: Alan J Husband, Department of Veterinary
Anatomy and Pathology, Building B14, University of Sydney, NSW
2006, Australia. Email: <a.husband@vetp.usyd.edu.au>
*
Present address: Novogen Ltd, 140 Wicks Road, North Ryde,
NSW 2112, Australia.
Received 12 March 1999; accepted 27 May 1999.
of isolated glucans, arising as a result of the original source
of material and method of preparation, influence biological
activity are unclear. Structural features hypothesized to
affect activity include molecular weight (MW), degree of
side-branching, helical conformation, solubility and gel-
forming ability.19 The greatest challenge to the development
of a therapeutic glucan is understanding the relationship
between the chemical structure and immunomodulatory
effects of these molecules so that different glucans can be tar-
geted to particular applications. The aim of this study was to
examine the temporal and dose-related effects on cells of the
peritoneal cavity, particularly with respect to macrophage
function, of glucans varying in MW and (1,6)-linkages, pre-
pared by alterations to the extraction procedure.
Materials and Methods
(1,3)-β-D-glucans
Yeast glucans differing in MW and proportion of (1,6)-linkages were
prepared by Novogen Ltd (North Ryde, NSW, Australia) from Sac-
charomyces cerevisiae, according to proprietary methodology. The
structural features of the compounds used are listed in Table 1.
Glucans were dissolved in DMSO prior to MW determinations using
gel permeation chromatography with dextran standards. Nuclear
magnetic resonance analysis was used to confirm that all compounds
contained a primary chain consisting of β-1,3-linkages and to deter-
mine the degree of β-1,6-linkages in the side-chains. All glucans
were suspended in endotoxin-free sterile water (Astra Pharmaceuti-
cals, North Ryde, NSW, Australia) prior to injection into mice.
All other reagents were obtained from Sigma Aldrich (Castle
Hill, NSW, Australia), unless stated otherwise.
396 JA Cleary et al.
Table 1 Structural characteristics of the glucans injected i.p.
Glucan Molecular weight β-(1,6)-
linkages
Low MW < 0.5 × 106 < 5%
Medium MW 0.5–2 × 106 < 5%
High MW > 4 × 106 < 5%
High MW, high (1,6)-linkages > 4 × 106 > 20%
Mice
Female, 6–10-week-old QS strain mice raised under specific
pathogen-free (SPF) conditions were kept in a PC2 animal house
facility for the duration of experiments, according to a University of
Sydney Animal Care and Ethics Committee approved protocol. All
control and treatment groups consisted of five mice/group unless
otherwise indicated.
Experimental design
To assess the kinetics of glucan action, mice were weighed and
injected, i.p., with one of the glucan preparations (0.2 mL, 5 mg/mL
prepared in water) at 12, 9, 6 or 3 days prior to assay. Blood, peri-
toneal cells and tissue samples were collected for assay from mice
anaesthetized with Penthrane (Abbott Laboratories, Abbott Park, IL,
USA). The effect of dose rate was determined 3 days after a single
i.p. injection of endotoxin-free sterile water (0.2 mL control mice) or
one of the glucan preparations (0.2 mL, 0.25–5 mg/mL). A compar-
ison of compound efficacy and the long-term effects of glucan were
examined 3, 28 or 90 days after injection of each glucan (0.2 mL, 1
mg), and compared with controls, which received endotoxin-free
sterile water (0.2 mL).
Sample collection
Blood was collected from anaesthetized mice by cardiac puncture
before they were killed. Blood was stored at 4°C overnight before
collecting serum by centrifugation.
Cells were collected from the peritoneal cavity by lavage of killed
mice with 4 mL sterile ice-cold PBS (pH 7.2) containing 5 U/mL
heparin sodium (CSL, Parkville, Vic., Australia). Lavage fluid was
kept on ice after the addition of 5 mL RF-10-PR media Roswell Park
Memorial Institute (RPMI)-1640 phenol red free-media containing
10% FCS (CSL), 2 mmol/L glutamine (ICN, Costa Mesa, CA, USA)
and 50 U/mL penicillin or 50 µg/mL streptomycin (Gibco BRL,
Grand Island, NY, USA) until centrifugation (300 g, 5 min, 4°C).
The cell pellet was resuspended in RF-10-PR media and the
number of macrophages was determined by counting cells on a
haemocytometer. Samples were adjusted to a concentration of
1 × 106 macrophages/mL. Cell viability was > 95%, determined by
trypan blue exclusion on random samples.
Differential cell counts
Before addition of RF-10-PR media, cells from peritoneal lavage
fluid (100 µL) were deposited as a pellet on glass slides using a
Shandon Cytospin 2 (150 g, 5 min; Shandon Southern Products,
Cheshire, England). Slides were air-dried, fixed in methanol and
stained with Diff-Quik (Lab Aids Pty Ltd, Narrabeen, NSW, Aust-
ralia). Five hundred cells per sample were identified by light
microscopy using a 100× oil-immersion objective to determine the
percentage of each cell type.
Macrophage morphology
The morphology of macrophages was examined by allowing adher-
ence of cells from peritoneal lavage fluid (60 µL) to sterile slidewells
(Lab-Tek, Nalge Nunc International, Naperville, IL, USA) for 2 h.
Cells were washed twice with warm RF-10-PR media, air-dried,
stained with Diff-Quik and observed by light microscopy.
Macrophage intracellular acid phosphatase assay
Freshly collected peritoneal lavage fluid (40 µL, containing
1 × 106 macrophages/mL) was incubated for 1.5 h at 37°C in flat-
bottomed, 96-well plates, then washed twice with warm RF-10-PR
media to leave purified macrophages adhered to the plate. The adher-
ent cells were lysed by addition of Triton-X (0.1%, 10 µL/well; ICN).
The pH was adjusted by addition of sodium acetate buffer
(0.2 mol/L, pH 5.0, 20 µL/well) and the substrate ρ-nitrophenyl
phosphate disodium salt (24 mmol/L prepared in sodium acetate
buffer pH 5.0, 20 µL/well; ICN) was added. The plate was incubated
for 30 min at 37°C and the reaction stopped by the addition of
sodium carbonate buffer (0.2 mol/L, pH 9.8, 200 µL/well).
Absorbance at 405 nm was determined on a SpectraMax plate reader
(Molecular Devices, Sunnyvale, CA, USA) and absolute concentra-
tions of acid phosphatase/4 × 104 macrophages were determined
using a standard curve prepared with purified acid phosphatase.
Stimulation indices were calculated by dividing treatment group
values by control macrophage values.
Macrophage nitrite assay
Peritoneal lavage fluid (60 µL, containing 1 × 106 macrophages/mL)
was incubated for 1.5 h at 37°C in flat-bottomed, 96-well plates, then
washed twice with warm RF-10-PR media to leave purified
macrophages adhered to the plate. The RF-10-PR media was
replaced in each well with or without high MW glucan (500 µg/mL),
LPS (2 µg/mL; E. coli serotype 0111 : 84) or NG-monomethyl-L-
arginine, monoacetate salt (100 µmol/L, L-NMMA; Calbiochem-
Novabiochem, Alexandria, NSW, Australia), bringing the final
volume to 300 µL/well. The plates were incubated for 2 days at 37°C.
Measurement of intracellular acid phosphatase was performed using
the same methods as for fresh macrophages. Nitrite was measured
according to a published protocol.8 Briefly, fresh Griess reagent (1%
sulphanilimide, 0.1% napthylethylene diamine hydrochloride, 2.5%
phosphoric acid, 100 µL/well) was added to culture supernatant
(100 µL/well) in a 96-well plate and incubated for 10 min at room
temperature. Absorbance at 550 nm was determined using a Spectra-
Max plate reader and absolute nitrite concentrations were determined
using a standard curve prepared with sodium nitrite (0–50 µmol/L).
Stimulation indices were calculated by dividing the LPS-stimulated
nitrite values of the treatment groups by the LPS-stimulated nitrite
production of control macrophages.
Macrophage superoxide production
Peritoneal lavage fluid (150 µL, containing 1 × 106 macrophages/
mL) was incubated for 1.5 h at 37°C in flat-bottomed, 96-well plates,
then washed twice with warm RF-10-PR media to leave purified
macrophages adhered to the plate. RF-10-PR media (300 µL) was
added per well and cells were incubated overnight at 37°C. Cells
were washed once with RF-10-PR before assaying.
Cytochrome c (2 mg/mL prepared in sterile HBSS, 100 µL/well)
was added to all of the triplicates. To one well from each sample
either HBSS (100 µL), HBSS (80 µL) and PMA (10 µg/mL, 20 µL)
or HBSS (60 µL), PMA (10 µg/mL, 20 µL) and superoxide dis-
mutase (SOD, 8800 U/mL, 20 µL) was added. The plates were
 Yeast glucan primes macrophage function
incubated for 90 min in a SpectraMax plate reader, temperature con-
trolled at 37°C. Absorbance at 550 nm and 540 nm was determined
every 30 s and plate-reader software calculated Vmax values. Stimu-
lation indices were calculated by dividing the PMA-stimulated
superoxide values of the treatment groups by the PMA-stimulated
superoxide production of control macrophages.
Serum lysozyme assay
Serum (200 µL/well) and Micrococcus lysodeikticus (4 mg/mL in
phosphate buffer 0.1 mol/L, pH 6.2, 100 µL/well) were added to each
well of a flat-bottomed 96-well plate. Clearance of M. lysodeikticus
was determined by absorbance measurements at 550 nm every 8 s for
2 min on a Spectromax plate reader. Lysozyme concentration was
determined by comparison with a standard curve prepared using
crystalline chicken egg white lysozyme (0–8 µg).
Histology
Sections of liver and spleen were collected from control and glucan-
treated mice following peritoneal lavage and fixed in 10%-phosphate
buffered formalin. Sections (5 µm) were stained with haematoxylin-
eosin stain and examined by light microscopy using a 40× objective.
Statistical analysis
All data are presented as the mean ± SΕΜ. Assay results were
analysed by one-way analysis of variance. When P < 0.05, Tukey’s
pair-wise comparisons were performed, assuming a family error rate
of 0.05, to determine which groups were significantly different.
Due to experimental design, only intraexperimental and not inter-
Figure 1 Proportions of (a) neutrophils, (b) eosinophils, (c) mast cells and (d) macrophages and lymphocytes found in the peritoneal
cavity after injection of low MW (s), medium MW (,) or high MW (h) glucan or high MW, high (1,6)-linkage glucan (n). Results are
expressed as the mean±SEM (n = 5/group).
397
experimental statistical analyses were valid. Hence, the results of the
compound efficacy and long-term effects of glucan experiments are
not directly comparable with the time and dose–response experi-
ments for each glucan.
Results
Kinetics of action
Initial experiments were performed to determine the influ-
ence of MW or degree of (1,6)-linkages on the kinetics of
macrophage activation by particulate yeast glucans.
Three days after i.p. injection of all glucans there were
significant changes to the population of cells found in the
peritoneal cavity. While glucan increased the number of
macrophages found in the peritoneal cavity at 3 days, there
was actually a reduction in the proportion of macrophages
and lymphocytes due to the infiltration of eosinophils and
neutrophils at this time (Fig. 1). The proportion of mast cells
was also reduced 3 days after injecting glucan. The propor-
tion of neutrophils and eosinophils returned to control values
(≤ 3%) over time, suggesting only an acute, transient inflam-
matory response to the glucans. The proportion of mast cells
remained low (< 1%) as a proportion of total cells over 12
days. By day 9, the proportion of macrophages and lympho-
cytes had returned to values equivalent to controls (≥ 85%),
in parallel with the decline in neutrophil and eosinophil
numbers. Changes in the blood level of eosinophils and
neutrophils were not measured.
Peritoneal macrophages were examined morphologically
and biochemically to determine their degree of activation.
398 JA Cleary et al.

Morphologically, macrophages from glucan-treated animals
appeared more activated, as shown by increased spreading 3
days after glucan injection (Fig. 2b) and increased rounding
and vacuolation after 6 days (Fig. 2c), compared with control
macrophages (Fig. 2a). Morphology slowly returned to
normal over time, although the rate at which this occurred
varied greatly between individual mice within the same
group. Twelve days after glucan injection, some samples still
contained a significant number of macrophages demonstrat-
ing an activated morphology (Fig. 2e), while activated cells
were absent in other samples (Fig. 2f).
Glucan injection resulted in a significant increase in
macrophage intracellular acid phosphatase activity, which
peaked at 3 days and then declined over time (Fig. 3).
There was no increase in basal nitrite production of
glucan-treated macrophages cultured for 2 days, compared to
control macrophages. In vitro stimulation with glucan for 2
days caused no increase in the nitrite production of either
control or in vivo glucan-treated macrophages. Stimulation
with LPS in vitro was only performed in the kinetic experi-
ments with macrophages from mice treated with high-MW,
high-linkage glucan. These cells produced substantially more

Figure 2 Typical morphology of macrophages collected from the peritoneal cavity of (a) control mice or (b) 3, (c) 6, (d) 9 or (e, f) 12
days after injection with medium MW glucan. Macrophages were incubated for 90 min at 37°C on glass slides.
 Yeast glucan primes macrophage function 399

nitrite in response to LPS than macrophages from control Comparison of compound activity
mice (48 ± 8 µmol/L compared with 5 ± 2 µmol/L, respec-
Comparison of compound efficacy was made 3 days after
tively, 3 days after injection of glucan) and the response was
same-day injection of all glucans.
maximal 3 days after administering the glucan.
All glucans stimulated morphological changes in the
In all the experiments, administration of glucan did not
macrophages, but distinct differences between the effects of
alter serum lysozyme concentration compared to control mice.
each glucan were not apparent. A statistically significant
Histological examination of liver sections from control and
reduction in the proportion of mast cells and an increased
glucan-treated animals revealed a non-significant increase in
proportion of neutrophils and eosinophils resulted after
inflammatory cell infiltrates in the livers of glucan-treated
administration of all glucans compared with controls
animals, which peaked 6 days after injection of glucan.
(Table 2), but there were no significant differences between
different glucans. However, there was a trend for an increased
Dose–response effect on macrophage activation neutrophilia associated with glucans of higher molecular
weight or increased (1,6)-linked branches.
The effect of dose on the ability of glucans to stimulate
Acid phosphatase activity was stimulated after injection
macrophages was assessed 3 days after injection, because this
of all glucans compared with controls and the higher molec-
was determined to be the day of greatest macrophage activa-
ular weight glucans had the greatest effect on this activity
tion in the kinetic experiments described earlier.
(Table 2). Medium MW and high MW glucans stimulated
For all glucans, acid phosphatase activity was markedly
significantly more acid phosphatase than the low MW
increased up to the 250 µg dose (Fig. 4a). With the exception
glucan, but this effect was negatively influenced by increased
of the medium MW glucan, no significant additional
(1,6)-linkages, because the high MW, high (1,6)-linked
enhancement of acid phosphatase activity was provided by
material was not as stimulatory as the high MW, low (1,6)-
increasing the dose to 1 mg. As in the kinetic experiments,
linked glucan.
basal nitrite production in the presence or absence of glucan
in vitro was low and no differences were observed between
macrophages from control and glucan-treated mice. In vitro,
stimulation with LPS increased nitrite production from
control and glucan-treated macrophages; however, this
increase was greater in the latter group and was directly
related to the in vivo dose of injected glucan (Fig. 4b).
Basal superoxide production by glucan-treated macro-
phages was low and no different to that of controls when
measured using the cytochrome c reduction assay. In contrast,
the response of glucan-treated macrophages after PMA
stimulation in vitro was a marked increase in superoxide
production compared with controls, which was related to
in vivo dose of glucan (Fig. 4c).
Glucan administration at any dose had no significant
effect on serum lysozyme concentrations, supporting the lack
of effect observed in the kinetic experiments. There was a
non-significant, dose-related increase in inflammatory infil-
trates in the livers associated with administration of glucan.

Figure 4 Stimulation indices for macrophage intracellular acid

Figure 3 Stimulation index of intracellular acid phosphatase
over time after injection of low MW (s), medium MW (,) or
high MW (h) glucan or high MW, high (1,6)-linkage glucan (n)
compared with macrophages from control mice (stimulation
index = 1). Results are expressed as mean±SEM (n = 5/group).
phosphatase (a), LPS-stimulated nitrite (b) and PMA-stimulated
superoxide production (c) in vitro after injecting increasing doses
of low MW (s), medium MW (,) or high MW (h) glucan or
high MW, high (1,6)-linkage glucan (n) compared with
macrophages from control mice (stimulation index = 1). Results
are expressed as the mean±SEM (n = 5/group).
400 JA Cleary et al.

Table 2 Peritoneal macrophage stimulation indices for intracellular acid phosphatase, nitrite (NO2) and superoxide (O2–) production, serum
lysozyme, % of each peritoneal cell type and liver histology 3 days after injection

Assay Controls Low MW Medium MW High MW High MW, high (1,6)-linkages

Acid phosphatase 1.00 ± 0.04 1.78 ± 0.07* 2.08 ± 0.15*† 2.18 ± 0.12*† 1.92 ± 0.14*§
Unstim. NO2 1.00 ± 0.25 0.44 ± 0.22 0.00 ± 0.10* 0.00 ± 0.11* 0.53 ± 0.32
LPS-stim. NO2 1.00 ± 0.07 2.64 ± 0.15* 2.85 ± 0.37* 2.68 ± 0.37* 3.74 ± 0.21*§
Unstim. O2– 1.00 ± 0.05 0.87 ± 0.03 0.95 ± 0.07 0.93 ± 0.08 1.03 ± 0.06
PMA-stim. O2– 1.00 ± 0.23 1.33 ± 0.11 1.87 ± 0.17* 1.78 ± 0.17* 1.85 ± 0.23*
Serum lysozyme 1.74 ± 0.14 1.76 ± 0.10 2.15 ± 0.13 2.13 ± 0.17 1.76 ± 0.10
Diff. Cell counts:
% Mast cells 3.6 ± 0.3 0.2 ± 0.1* 0.1 ± 0.0* 0.1* ± 0.1 0.2 ± 0.0*
% Neutrophils 0.3 ± 0.2 24.4 ± 4.3* 21.1 ± 3.5* 26.2 ± 3.3* 31.1 ± 6.0*
% Eosinophils 1.1 ± 0.6 7.0 ± 1.5* 12.5 ± 1.8* 11.2 ± 1.0* 7.2 ± 1.4*
% Mac. + Lymph. 95.0 ± 0.6 68.3 ± 5.0* 66.3 ± 2.9* 62.6 ± 2.8* 61.6 ± 4.9*
Liver histology:
< 20cells/lesion 1.14 ± 0.77 3.08 ± 0.8 3.00 ± 0.74 2.13 ± 0.88 1.57 ± 0.34
20–50cells/lesion 0.00 ± 0.00 0.31 ± 0.16 1.64 ± 0.79 0.57 ± 0.35 1.16 ± 0.29
> 50cells/lesion 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.11 ± 0.11 0.00 ± 0.00

Data shown are the mean ± SEM; n = 5/group. Serum lysozyme is measured in µg/mL and liver histology as lesions/100 fields-of-view (40×).
*
P < 0.05 compared with controls; †P < 0.05 compared with low MW; §P < 0.05 compared with high MW. Unstim., unstimulated; stim., stimu-
lated; Diff., differential; Mac., macrophages; Lymph., lymphocytes.

Basal nitrite production by in vivo glucan-treated peri-
toneal macrophages was reduced (significantly, in the case of
the medium and high MW glucans) compared with controls
(Table 2). After LPS stimulation, however, macrophages from
all glucan-treated macrophages produced significantly more
nitrite than control macrophages. There was no significant
difference between glucans of differing MW, but increasing
the proportion of (1,6)-linkages significantly increased the
nitrite production in response to LPS in vitro.
Unstimulated superoxide production from macrophages
of all glucan-treated mice was no different to control
macrophages, but the response to PMA was enhanced after
glucan administration, especially with the higher MW
glucans (Table 2). Increasing the percentage of (1,6)-linkages
did not have any effect above that of increasing the MW of
the glucan injected.
As found previously, serum lysozyme concentrations
were not affected after the administration of any glucan
(Table 2).
Liver histology was not significantly altered after glucan
administration, although the livers from glucan-treated mice
tended to have increased inflammatory cell infiltrates com-
pared to livers from control mice (Table 2).
Long-term effects
Macrophage function was assayed 28 and 90 days after i.p.
injection to determine whether treatment with glucan exerted
any long-term effects.
Control and glucan-treated peritoneal populations had
essentially the same proportion of macrophages, lymphocytes
and eosinophils 28 and 90 days after injection of glucan.
There was a slight non-significant increase in neutrophils 28
days after injection of the higher MW and (1,6)-linkage
glucans, but these had returned to normal after 90 days. Mast
cell populations remained significantly reduced in all glucan-
treated groups after 28 days, independent of structural dif-
ferences, with the proportion of mast cells only resembling
control values after 90 days.
Intracellular acid phosphatase levels of control and
glucan-treated macrophages were not different 28 or 90 days
after injection of glucan (Table 3) with the exception of the
medium MW group, in which acid phosphatase levels were
significantly raised compared with the control and high MW,
high (1,6)-linkage glucan groups at day 90.
Peritoneal macrophages from control and glucan-treated
mice generally had analogous basal and in vitro glucan-stim-
ulated levels of nitrite production at 28 and 90 days. The
exceptions were the high MW, high (1,6)-linked glucan
group, which showed significantly increased nitrite produc-
tion at day 28, and the medium MW glucan group, which
showed an increase at day 90, compared with all other
groups. Overall, macrophages from glucan-treated mice pro-
duced marginally more nitrite in response to LPS than control
macrophages, with a statistically significant increase com-
pared to the controls for the high MW, high (1,6)-linked
glucan at day 28 and the medium MW glucan at day 90
(Table 3).
After 28 and 90 days, basal superoxide production was no
different between all treatment groups. Only at day 28 was
there a significant increase in PMA-stimulated superoxide
production from the medium MW and high MW, high (1,6)-
linked glucans. There was no difference between the effec-
tiveness of the different glucans, indicating that the MW and
degree of (1,6)-linkages did not influence superoxide pro-
duction (Table 3).
There was no difference in serum lysozyme between
control and glucan-treated mice at either 28 or 90 days after
treatment.
Inflammatory infiltrates were more commonly observed
in the livers of glucan-treated mice, compared with control
 Yeast glucan primes macrophage function
Table 3 Stimulation indices for macrophage acid phosphatase, LPS-stimulated nitrite (NO2) and PMA-stimulated superoxide (O2–)
production in vitro, 28 or 90 days after injection
Assay Glucan
Acid Phosphatase Controls
Low MW
Medium MW
High MW
High MW, high (1,6)-links
LPS-stim. NO2 Controls
Low MW
Medium MW
High MW
High MW, high (1,6)-links
PMA-stim. O2– Controls
Low MW
Medium MW
High MW
High MW, high (1,6)-links
Data shown are the mean ± SΕΜ. n = 5/group for 28 days and n = 6/group for 90 days. *P < 0.05 compared with controls; †P < 0.05 compared
with low MW; ‡P < 0.05 compared with medium MW; §P < 0.05 compared with high MW. Stim., stimulated.
mice, at both 28 and 90 days. The increase was only signifi-
cant for infiltrates containing < 20 cells/lesion in the medium
MW and high MW, high (1,6)-linked material at 90 days
compared with controls.
Discussion
In the present study, injection of glucan into the peritoneal
cavity altered both the morphology and activity of peritoneal
macrophages. This response was both time-dependent and
dose-dependent and is presumably due to both a direct effect
of glucan on the macrophages and an indirect effect via its
influence on surrounding cells.
After injection of glucan, neutrophils and eosinophils
infiltrated the peritoneal cavity and mast cells numbers were
reduced, but the sequence of events was unclear. This reduc-
tion in the proportion of mast cells has not previously been
reported. Direct interaction of glucan with macrophages
could induce the release of mediators that cause mast cell
degranulation or trafficking away from the peritoneal cavity
and neutrophil/eosinophil accumulation. Alternatively,
glucan may interact directly with the mast cells. If mast cell
degranulation occurs, the release of preformed TNF-α may
enhance chemotaxis of neutrophils20 as well as mediators
released by resident cells. Instead, neutrophils and
eosinophils may be attracted to the site by other chemokines
and it could be the release of mediators by these accumulat-
ing cells, rather than or in addition to macrophages, that
induce the changes observed in the mast cell population.
Whatever the exact sequence of events, the subsequent milieu
of cells and soluble factors found in the in vivo micro-envi-
ronment after injection of glucan provides additional stimu-
latory signals to the macrophages that are not provided by
their in vitro interaction with glucan. For example, in vitro,
glucan and IFN-γ have separate marginal effects on the secre-
tion of macrophage products. However, when in combination,
401
28 days 90 days
1.00 ± 0.06 1.00 ± 0.06
0.99 ± 0.12 1.08 ± 0.05
0.99 ± 0.12 1.26 ± 0.08*
0.97 ± 0.11 1.03 ± 0.17‡
0.92 ± 0.14 1.12 ± 0.13
1.00 ± 0.33 1.00 ± 0.10
0.89 ± 0.33 1.68 ± 0.59
1.43 ± 0.24 3.80 ± 0.72*†
1.80 ± 0.74 1.90 ± 0.62‡
3.38 ± 0.89*†§ 1.86 ± 0.35‡
1.00 ± 0.06 1.00 ± 0.12
1.81 ± 0.27 0.61 ± 0.08
2.55 ± 0.36* 0.82 ± 0.16
1.61 ± 0.23 0.91 ± 0.07
1.87 ± 0.19* 0.87 ± 0.11
glucan and IFN-γ cause macrophage activation similar to that
observed following in vivo stimulation with glucan alone.8,21
The change in morphology of glucan-treated macrophages
observed in the present study is consistent with the litera-
ture.6 Activated macrophages typically show increased mem-
brane ruffling, increased adhesion and spreading, induction
of DNA synthesis, altered monokine secretion, increased
lysosomal enzyme levels, altered phagocytic activity and
increased bactericidal/tumouricidal activity. After binding to
macrophages, particulate glucans are phagocytosed and
subsequent signalling mechanisms, which have not yet been
elucidated, stimulate macrophage activity. The vacuolation
(Fig. 2) indicates the stimulation of macrophage phagocyto-
sis and increased ruffling and spreading suggests activation
of macrophages by glucan.
Glucan administration increased the levels of the
macrophage lysosomal enzyme acid phosphatase and the
nitrite and superoxide response of macrophages in response
to a second inflammatory signal. The increase in these activ-
ities leads to the enhanced bactericidal/tumoricidal properties
of macrophages and could explain the ability of glucan-
treated animals to more readily clear infection or inhibit
tumour growth than untreated animals.1,3 Because glucan
only enhanced nitric oxide and superoxide release after a sec-
ondary signal, this suggests that glucan primes macrophage
function without stimulating increased basal activity, thereby
preventing damaging inflammatory processes. Although
there was an acute inflammatory response to glucan, as indi-
cated by the influx of neutrophils and eosinophils into the
peritoneum, this was transient and the priming effect on
macrophages outlived this acute response.
Prior to performing in vitro assays to assess the degree of
macrophage activation, macrophages were purified by adher-
ence from peritoneal lavage samples adjusted to the same
macrophage concentration. It was assumed that macrophages
from control and glucan-treated mice were equally adherent
and no estimation of cell number attached to the 96-well
402 JA Cleary et al.
plates after adherence was made. We cannot rule out the
possibility that the increased acid phosphatase activity and
stimulated nitric oxide and superoxide production of macro-
phages from glucan-treated mice was not merely due to an
increase in cell number in those wells. However, increased
adherence in itself could be used as an indication of
macrophage activation and would not contradict the findings
reported above.
Lysozyme is a bactericidal enzyme constitutively released
by macrophages. Lysozyme production is increased when the
cells are activated. If released in close proximity to invading
microbes it can enable digestion of their cell walls. In the
present study, serum lysozyme was used as an indicator of
activation of systemic immune function following i.p. deliv-
ery of glucan. As no change in serum lysozyme was
observed, this suggests local activation of peritoneal cells
rather than systemic immune activation. This may be due to
particulate glucan being too large to diffuse out of the peri-
toneal cavity or, alternatively, it may be efficiently trapped by
the cells within the peritoneal cavity that remain localized at
that site, inducing very little systemic response.
The role of a cell-surface receptor for glucan remains con-
troversial. Czop has identified a specific glucan receptor that
exists on human monocytes, neutrophils and murine macro-
phages.22 The smallest ligand for this receptor is only 7
glucose units long.23 Alternatively, the complement receptor,
CR3 (CD11b/CD18), may be bound because it has a glucan-
binding domain on the CD11b molecule.14 Binding to the
latter receptor may explain why glucan has been reported to
affect the function of cells other than macrophages, including
B and T cells, neutrophils, eosinophils and NK cells, all of
which have the CR3 receptor. The role of glucan in activat-
ing cells other than macrophages should not be ignored.
Structural features that are reported to be important for
biological efficacy of glucans include the MW, degree of
(1,6)-linked side-branching, helical conformation and solu-
bility. For example, MW alone is reported to influence acti-
vation of the alternate complement pathway,12 macrophage
tumour necrosis factor-α (TNF-α)24,25 and superoxide pro-
duction,26 macrophage phagocytic activity27 and blood clear-
ance of colloidal carbon.28 However, the reported effects of
structure are often contradictory, probably due to testing of
glucans from different sources, isolated in different ways and
tested either in vitro or in vivo. For example, Okazaki et al.
have suggested that TNF-α production is greatest after in
vitro stimulation with glucans having high MW and low
branching ratios and that the helical conformation of gel-
forming glucans is unimportant for TNF-α production.24 In
contrast, Kulicke et al. have reported that low MW glucan
stimulates higher TNF-α release in vitro than the higher MW
molecules and that helical conformation does influence TNF-
α production.25 Ohno et al. have found that high MW, high
branching and helical ultrastructure of the glucan are all
important for in vivo priming of macrophages for LPS-stim-
ulated TNF-α release.29 Not only does the source of the
glucan influence its structure, but the method of isolation can
also significantly affect the final product.30 The present study
attempted to reduce the unknown influence of source mater-
ial on chemical structure by comparing glucans obtained
from one yeast species and modifying the chemical structure
via the isolation procedure alone.
Our studies have demonstrated that all the glucans tested
stimulate macrophage function above that of nonelicited,
control peritoneal macrophages. Varying the molecular
weight and side-branching of the glucans did not signifi-
cantly change the degree of macrophage activation, although
there was a tendency for glucans with higher molecular
weight and (1,6)-linkages to be more stimulatory. All the
glucans tested were much larger than the heptaglucoside that
has been shown to bind the glucan receptor. Therefore, the
lack of any significant difference between the activity of
glucans with different structural attributes may be due to an
inability of the receptor to differentiate between molecules in
the MW range selected. It has also been suggested that stim-
ulation of some cellular activities requires receptor cross-
linking.31 If the higher MW or more highly (1,6)-linked
materials were better able to cross-link receptors, this may
account for the observed trends in glucan activity. The degree
of (1,6)-linkages appears to have a stronger influence than
MW on the extent of macrophage activation, which may also
be related to the ability to cross-link receptors. However, the
impact of other structural features such as helical conforma-
tion, which may also influence the biological efficacy of
these compounds, were not assessed.
To date, the clinical application of glucan has focused on
the use of soluble derivatives of glucan, because of their ease
of delivery and the implication that intravenous administra-
tion of particulate glucan induces severe inflammatory
responses, such as granuloma formation.32–34 While the dif-
ferential cell counts of the populations found in the peritoneal
cavity suggest an acute, transient inflammatory response, his-
tological examination of liver sections of mice injected, i.p.,
with particulate glucan in these studies revealed no signifi-
cant increase in granuloma formation up to 90 days post-
injection. This implies a lack of toxicity after i.p. delivery,
which supports the literature on topical35 and intralesional36
delivery of insoluble glucan. Taking these studies together,
it can be speculated that particulate glucan localizes at the
site of delivery, invoking a local rather than systemic
immunomodulatory effect as indicated by the absence of any
alteration to serum lysozyme levels in the present study.
Hence, particulate glucan may be most effective when a local
rather than systemic enhancement of immune function is
required.
The experiments discussed earlier were performed only
once following the exact protocol described. However,
similar experiments where different combinations of the
glucans with altered structure were tested gave qualitatively
the same results, suggesting that the trends described are
reproducible. Future studies will examine the efficacy of the
immunomodulatory impact of glucan in models of infection
and cancer. This will further clarify the importance of
priming macrophage activity for enhancing the host’s ability
to maintain a healthy immune environment.
Acknowledgements
Janelle Cleary was the recipient of an Australian Postgradu-
ate Award (Industry) and this work was supported in part by
Novogen Limited. The technical assistance of Phillipa Zucker
and proofreading by Dr Wendy Muir was greatly appreciated.
 Yeast glucan primes macrophage function
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