Microbial. Immunol., 41(12), 991-998, 1997

Immunomodulatory Activities of Oat ƒÀ-Glucan

In Vitro and In Vivo

Alberto Estrada*,1, Cheol-Heui Yun, Andrew Van Kessel, Bing Li, Shirley Hauta,

and Bernard Laarveld

Animal Biotechnology Centre, Department of Animal and Poultry Science, University of Saskatchewan, 72 Campus Drive, Saska-toon, Saskatchewan, Canada S7N 5B5

Received July 29, 1997. Accepted September 25, 1997

Abstract: Previous studies have shown that ƒÀ-glucans extracted from yeast or fungi potentiate immune

responses. In the present study, the immunomodulatory activities of 13-(1•¨3, 1•¨4)-glucan, derived from

oats, were investigated. The ability of oat ƒÀ-glucan (OƒÀG) to stimulate IL-1 and TNF-a release from murine

peritoneal macrophages and the murine macrophage cell line P338D1, was assessed. In vitro stimulation of

macrophages with OƒÀG resulted in the production of IL-1 in a dose and time-dependent manner, where-

as only small amounts of TNF-a could be detected in the culture supernatants. OƒÀG also induced the pro-

duction of IL-2, IFN-ƒÁ and IL-4 secretion in a dose-dependent manner in cultured spleen cells. The

intraperitoneal administration of OƒÀG in mice resulted in the accumulation of leucocytes, predominant-

ly macrophages, in the peritoneal cavity. Furthermore, OƒÀG was tested for its ability to enhance non-spe-

cific resistance to a bacterial challenge in mice. Survival of mice challenged with Staphylococcus aureus was

enhanced by a single intraperitoneal administration of 500 ƒÊg of OƒÀG 3 days prior to bacterial challenge.

In conclusion, these studies demonstrated that OƒÀG possesses immunomodulatory activities capable of stim-

ulating immune functions both in vitro and in vivo.

Key words: Oat ƒÀ-glucan, Immunomodulation

β-glucans are homopolymers of D-glucose and majorstructural components of the cell wall of yeast, fungi and

some cereals such as barley and oats. The cell wall

glucans of yeast and fungi consist of two structurally dis-

tinct components of 1•¨3-linked glucopyranosyl residues

with small numbers of 1•¨6-linked branches (21). ƒÀ-glu-

can with 1•¨3 and 1•¨4 linkages is present in the oat

endosperm cell walls as a polysaccharide structure of

approximately 2 •~106 molecular weight (49). 13C-nuclear

magnetic resonance (NMR) spectrum for oat 13-glucan

indicates that 1•¨3 linkages occur singly, whereas most

of the 1•¨4 linkages occur in groups of two or three. The

resultant structure for oat ƒÀ-glucan is a linear, unbranched

polysaccharide composed mainly of ƒÀ-1•¨3-linked cel-

lotriosyl and cellotetraosyl units (48). Oat and barley ƒÀ-

glucan have been generally categorized as structurally

identical (18). No X-ray crystallographic studies have

been reported for oat ƒÀ-glucan, but barley 13-glucan was

characterized as a three-fold helix with three cellotriose

units per turn (40).

β-glucans are known to act as adjuvants (3, 17) and asimmunostimulants (2, 19, 20, 24, 37, 47). 13-glucans

enhance the activities not only of macrophages (20, 26),

but also of neutrophils (43), natural killer (NK) cells

(11, 43), T cells (14, 30) and B cells (14, 35). As vaccine

adjuvants, ƒÀ-glucans potentiate humoral and cellular

immune responses to a diverse group of microbial and

tumor antigens. They also non-specifically enhance the

host resistance to neoplastic (36), bacterial (15, 19, 27),

viral (41, 45, 46), protozoan (13) and fungal (4) dis-

eases. This broad spectrum of activities may be pri-

marily attributed to macrophage activation. In vivo and

in vitro studies have shown that ƒÀ-glucans increase

macrophage phagocytosis (33) and macrophage cytokine

production (15, 16, 23, 31).

One mode of action of ƒÀ-glucans involves mono-

cyte/macrophage stimulation and release of mediators

involved in inflammation like prostaglandin E2, inter-

* Address correspondence to Dr . Alberto Estrada, Animal

Biotechnology Centre, Department of Animal and Poultry Sci-

ence, University of Saskatchewan, 72 Campus Drive, Saskatoon,

Saskatchewan, Canada S7N 5B5. E-mail: estrada@SASK.USASK. CA

Abbreviations: CFU, colony forming units; FCS, fetal calf

serum; IFN-ƒÁ, gamma interferon; IL, interleukin; IP, intraperi-

toneal; OƒÀG, oat ƒÀ-glucan; PBS, phosphate buffered saline;

TNF-ƒ¿, tumor necrosis factor alpha.

99 1

99 2 A. ESTRADA ET AL

leukin-1 (IL-1) and tumor necrosis factor alpha (TNF-a)

(26). It is likely that the stimulation of monocytes/macro-

phages is mediated by the binding of the ƒÀ-glucans to

their specific receptors on these cells (19). Human (6)

and murine (12, 19) macrophages possess receptors with

specificity for ƒÀ-glucans with 1•¨3 linkages, and it is

probable that most mammalian species display similar

receptors. Studies have demonstrated that ƒÀ-glucan

from barley recognizes the specific ƒÀ-glucan receptors on

human macrophages (5). Although no studies of this

nature have been carried out with ƒÀ-glucan extracted

from oats, it is probable that this glucan binds the same

receptors on macrophages due to its identical chemical

structure to barley ƒÀ-glucan. ƒÀ-(1•¨3, 1•¨4)-glucan

derived from oats was previously demonstrated to have

immunostimulatory effects as observed by the enhanced

disease resistance of immunosuppressed mice to Eimeria

vermiformis infection (50). The objective of the present

study was to investigate the immunological effects of oat

β-glucan (0ƒÀG) on immune responses in vitro and in

vivo. This study indicates the potential of 0ƒÀG as an

immunostimulant.

Materials and Methods

Reagents. ƒÀ-(1•¨3,1•¨4 glucan prepared

from oat endosperm was obtained from Ceapro Inc.

(Edmonton, AB, Canada). The endotoxin contamination

of this preparation was less than 10 pg/mg as deter-

mined by a Limulus amebocyte lysate chromogenic spe-

cific assay (BioWhittaker, Inc., Walkersville, Md., U.S.A.).

The molecular weight of 0ƒÀG was 1.1 X 106 daltons as

determined by a TSK-60 high-performance liquid chro-

matography (HPLC) column (Bio-Rad, Mississauga,

ON, Canada) according to the method described by

Wood et al (49) for the molecular characterization of

cereal ƒÀ-glucans. 0ƒÀG was soluble at a concentration up

to 20 mg/ml. Zymosan was purchased from Sigma

Chemical Co. (St. Louis, Mo., U.S.A.). Precautions

were taken to eliminate endotoxin contamination in the

experiments; all glassware was washed and rinsed with

deionized, double-distilled water and sterilized by heat-

ing at 175 C for 3 hr. All media and solutions used for

cell cultures and inoculations were pyrogen-free and

tested by the Limulus assay before use.

Animals. CD1 (Animal Resources Centre, Universi-

ty of Saskatchewan, Saskatoon, SK, Canada) or BALB/c

mice (Charles River Laboratories, St-Constant, Quebec,

Canada), 6 to 8 weeks of age, were used. The experi-

mental protocols used were approved by the Animal

Care Committee of the University of Saskatchewan and

were performed in accordance with the recommendations

of the Canadian Council on Animal Care as specified in

the Guide to the Care and Use of Experimental Ani-

mals.

In vitro studies: Macrophage cultures. Peritoneal

macrophages were harvested by washing the peritoneal

cavity of five BALB/c mice with 5 ml of cold RPMI with

gentle massage to dislodge any loosely adherent cells.

Lavaged cells were centrifuged at 300 X g for 10 min and

pooled. Pooled cells were washed once with RPMI-

10% FCS. The cells were counted and adjusted to 1 X

106 cells/ml. Macrophage monolayers were established

by seeding 2 X 105 cells/200 ill of RPMI-10% FCS in 96-

well flat-bottom cell culture microtiter plates (Corning

Glass Works, Corning, N.Y., U.S.A.), incubating them in

a 5% CO, atmosphere at 37 C for 4 hr, and washing them

with RPMI to remove non-adherent cells. The cell cul-

tures in RPMI-10% FCS were incubated at 37 C in a 5c7c

CO, atmosphere for 2 hr, at this time, the medium was

removed and replaced with 200ƒÊ1/well of RPMI-10k

FCS containing OPG or zymosan at concentrations from

10 to 1,000 ƒÊg/ml. The cultures were incubated for 3. 6.

24 or 48 hr and the supernatants taken at these time

points.

The P338D1 macrophage cell line ATCC TIB-63

(American Type Culture Collection, Rockville, Md..

U.S.A.) was cultured in 96-well microtiter plates at 1 X

105 cells per well in RPMI 1640 medium (RPMI.

GIBCO, Grand Island, N.Y., U.S.A.) containing 107c

fetal calf serum (FCS, GIBCO-RPMI-FCS) and incu-

bated at 37 C in a 5% CO, atmosphere. After 3 days in

culture, the medium was replaced with fresh RPMI-59

FCS containing 0ƒÀG or zymosan at concentrations from

10 to 1,000 ƒÊg/ml. The cells were further cultured for 2.

4 or 7 days for the determination of IL-1 and for 6, 24 or

48 hr for the determination of TNF-a and the super-

natants taken at these time points.

Determination of IL-1 and TNF-a cytokines in

macrophage culture supernatants. Enzyme-linked

immunoassay (ELISA) was used to measure IL-1 and

TNF-a cytokines in the macrophage culture super-

natants. Briefly, individual wells of 96-well microtiter

plates (Immuno Plate; Nunc, Inter-Med, Denmark) were

coated with monoclonal anti-mouse IL-1-a (Genzyme

Diagnostics, Cambridge, Mass., U.S.A.) or TNF-a anti-

bodies (Endogen, Cambridge, Mass., U.S.A.) in phos-

phate buffered saline (PBS). The wells were washed

with PBS buffer containing 0.05% Tween-20 (PBS-T).

Standard recombinant IL-1-a (Genzyme) and TNF-a

(Endogen) cytokine dilutions and macrophage culture

supernatant 1/2 to 1/8 dilutions in PBS-T were added to

the wells and incubated at 37 C for 2 hr (IL-1) or at 20 C

for 18 hr (TNF-a). The wells were then washed with

PBS-T and monoclonal biotinylated detecting antibodies

anti-IL-1-a (Genzyme) or anti-TNF-oc (Endogen) in

IMMUNOMODULATORY ACTIVITIES OF OAT [3-GLUCAN 993

PBS-T were added. The wells were washed with PBS-T and streptavidin alkaline phosphatase-conjugated (GIBCO) (IL-1) or streptavidin horseradish peroxidase-conjugated (Cedarlane Laboratories, Hornby, ON, Cana-da) (TNF-a) in PBS-T were added and incubated at 37 C for 1 hr. The plates were washed with PBS-T and the substrate solutions, p-nitrophenyl phosphate (Sigma)

(IL-1) or tetramethylbenzidine (Sigma) (TNF-a) were added. The absorbance of each well at 405 nm (IL-1) or 450 nm (TNF-a) was measured using an automated spectrophotometer (Molecular Devices V. Kinetic microplate reader; Molecular Devices, Menlo Park, Calif., U.S.A.). IL-1 and TNF-a concentrations in macrophage culture supernatants were determined by regression analysis using the optical density (OD) read-ings provided by the recombinant standard cytokines and the dilutions of the culture supernatants. The results were expressed as the IL-1 or TNF-a amounts per ml of culture supernatant.

Determination of IL-2, IFN- y and IL-4 cytokines in spleen cell culture supernatants. Lymphocytes were obtained from the spleen of BALB/c mice. Spleens from five mice were teased apart with forceps into RPMI-5% FCS and meshed through a 70 pm cell strain-er (Falcon, Becton Dickinson Labware, Franklin Lakes, N.J., U.S.A.). The cells were centrifuged at 300 X g at 4 C for 10 min and the cell pellet treated with 1% ammonium chloride solution to lyse erythrocytes. The cells were washed twice with RPMI-5% FCS by centrifugation, resuspended in RPMI-10% FCS, counted and the cell concentration adjusted to 2 X 106 cells/ml. One hun-dred microliters of isolated spleen cells were added to wells of 96-well flat-bottom cell culture microtiter plates.

OPG or zymosan, at concentrations from 5 to 800 lag/ml in 100 tl of RPMI-10% FCS, were added to wells in trip-licate cultures. The cultures were incubated for 48 hr and the supernatants were collected. ELISA was used to measure IL-2, IFN-y and IL-4 produced by the spleen cell cultures following the above indicated protocol for the detection of TNF-a. Monoclonal anti-IL-2, IFN-y or IL-4 antibodies (Endogen), and biotinylated anti-IL-2, TEN-)/ or IL-4 antibodies (Endogen) were used for coating and detection, respectively. Standard recombinant IL-2,

IFN-7 and IL-4 cytokines (Endogen) were used to deter-mine the cytokine concentrations in the spleen culture supernatants by regression analysis. The results were expressed as the amount of IL-2, IFN-7 or IL-4 per ml of culture supernatant.

In vivo studies: Determination of leucocyte numbers, cell kinetics and differential cell counts in the peritoneal cavity. Groups of five CD-1 mice were injected IP with 200 tl of PBS alone or PBS containing 500 pg of OPG or zymosan. Mice were euthanized by halothane inhala-

tion at days 1, 3, 5 and 10 after treatment and the cells

were obtained by injecting 5 ml of RPMI-10% FCS

into the peritoneal cavity. The total number of peri-

toneal cells per mouse was determined using a Coulter

Counter (Coulter Electronics, Inc., Hialeah, Fla., U.S.A.).

The cells were then adjusted to 1•~10°/ml in PBS and

100 pi of the suspension were placed on slides, which

were centrifuged at 500•~g for 4 min using a cytospin cen-

trifuge (Fisher Scientific Ltd., Nepean, ON, Canada).

The resultant smears were air dried at room temperature

and then fixed and stained by Leukostat solution (Fisher

Diagnostics, Pittsburgh, Pa., U.S.A.). The slides were

examined by light microscopy and differential counts of

100 leucocytes from each mouse sample were deter-

mined. The cells were differentiated into the following

categories: total leucocytes, lymphocytes, macrophages

and neutrophils. Leucocyte numbers were calculated by

the following formula:

Leucocyte No.

= (No. of cells per ml•~5)•~(% of leucocyte type)

100

The results were expressed as the mean total cell

numbers •} SEM.

Bacterial challenge of mice. Staphylococcus aureus

(ATCC-25923) was used as the challenge strain. Bacteria

were grown in trypticase soy broth (Becton Dickinson,

Cockeysville, Md., U.S.A.) at 37 C for 18 hr. The bacte-

ria were diluted as appropriate in fresh nutrient broth

immediately before challenge, and viable colony counts

were determined. Serial decimal dilutions were made

and 201,11 samples of each dilution were plated in tripli-

cate on nutrient agar to determine the viable cell densi-

ty for the inoculum. The bacterial colony counts were

reported as the logo colony forming units (CFU) per

ml. Groups of 10 CD1 mice were treated IP with 200 tl

of PBS alone or PBS containing 500 pg of OPG or

zymosan 3 days before challenge with S. aureus. Chal-

lenge was performed by the IP injection of 100 pi of bac-

terial suspensions diluted in sterile PBS containing 1 X

10', 5 X 109 or 5 X 108 CFU. The mice were observed

four times per day for the first 72 hr and twice per day

thereafter. Moribund animals were euthanized by

halothane inhalation. Clinical signs including mortality

were recorded. Mice alive 10 days after bacterial chal-

lenge were recorded as survivors.

Statistics. Results are expressed as means ± standard

error of the mean (SEM) and, when applicable, compared

by the Student's t-test. Comparison of groups with

regard to survival was made by chi-square analysis.

99 4 A. ESTRADA ET AL

Results

In Vitro Effects of OPG: 1L-1 and TNF-a Production

following OƒÀG Stimulation of Macrophages

The ability of 0ƒÀG to induce IL-1 and TNF-a release

by macrophages was evaluated using a primary culture of

peritoneal cells and the macrophage cell line P338D1.

Results in Fig. 1 show that 0ƒÀG stimulated the secretion

of IL-1 in a dose and time-dependent fashion in both pri-

mary cultures (Fig. la) and the macrophage cell line

(Fig. lb). The kinetics of IL-1 production were different

between the peritoneal macrophages and the P338D1 cell

line. Peritoneal macrophages reached maximal levels of

IL-1 secretion at 48 hr following stimulation with 0ƒÀG

or zymosan. In contrast, P338D1 cells required a longer

incubation time with the stimulants to produce similar

levels of IL-1 secretion. Generally, the stimulation of

macrophages with 013G induced higher IL-1 levels than

zymosan at the same doses or incubation times. Removal

of traces of endotoxin from the OƒÀG preparation using

immobilized polymyxin B did not lower subsequent

induction of IL-1 release from P388D1 cells (data not

shown).

Figure 2 illustrates the secretion of TNF-a by peri-

toneal and cell line macrophages in the presence of var-

ious amounts of 0ƒÀG or zymosan. From peritoneal

macrophages (Fig. 2a), 0ƒÀG induced TNF-cx release at 3

hr following stimulation, TNF-a levels increased by 6 hr

( a ) ( b )

and sharply declined at 24 hr in the culture supernatant.

All zymosan concentrations from 10 to 1,000ƒÊg/m1

induced the release of higher levels of TNF-a than 0ƒÀG

at 6 and 24 hr incubation. From cell line macrophages

(Fig. 2b), at 6 and 24 hr incubation, 0ƒÀG appeared to

induce minimal amounts of TNF-a which increased by

( a ) ( b )

IL-2 IFN-y IL-4

Fig. l . Dose and time effects of oat P-glucan (•œ) and zymosan

(•›) on the release of IL-I by macrophages. Primary cultures of

mouse peritoneal macrophages (a) and the murine macrophage

cell line P338D 1 (b) were co-cultured with different concentra-

tions of the stimulants. Culture supernatants were collected at the

indicated time points and tested for IL-I by ELISA. IL-I amounts

were calculated by a standard curve using recombinant murine IL-

1-ƒ¿. The data are expressed as the concentration of IL-1 in culture

supernatants in pg/ml. Each point represents the mean of triplicate

values ± SEM.

Fig. 2. Dose and time effects of oat ƒÀ-glucan (•œ) and zymosan

(•›) on the release of TNF-a by macrophages. Primary cultures

of mouse peritoneal macrophages (a) and the murine macrophage

cell line P338D1 (b) were co-cultured with different concentra-

tions of the stimulants. Culture supernatants were collected at the

indicated time points and tested for TNF-a by ELISA. TNF-a

amounts were calculated by a standard curve using recombinant

murine TNF-a. The data are expressed as the concentration of

TNF-a in culture supernatants in pg/ml. Each point represents the

mean of triplicate values ± SEM.

Fig. 3. Dose-dependent effect of oat ƒÀ-glucan (•œ) and zymosan

(•›) on the release of IFN-y, IL-2 and IL-4 by lymphocytes.

Mouse splenic lymphocytes were co-cultured with different con-

centrations of the stimulants for 48 hr. Culture supernatants were

collected at the indicatd time points and tested for IL-2. IFN-y

and IL-4 cytokines by ELISA. Cytokine amounts were calculat-

ed by a standard curve using recombinant murine IL-2, IFN-y and

IL-4. The data are expressed as the concentration of cytokines in

culture supernatants in pg/ml. Each point represents the mean of

triplicate values •} SEM.

IMMUNOMODULATORY ACTIVITIES OF OAT P-GLUCAN 99 5

48 hr, however, no dose effect was observed. In general,

the stimulation of macrophages with zymosan induced

higher TNF-a levels than OƒÀG at the same doses or

incubation times.

IL-2, IFN- y and IL-4 Secretion in Spleen Cell Culture

Supernatants

Previous observations have suggested that ƒÀ-glucans

derived from yeast and fungi are capable of stimulating

the release of cytokines that are mainly associated with

T and NK cells (29, 32). On these bases, the ability of

0ƒÀG to stimulate the secretion of cytokines such as IL-

2. IFN-y and IL-4 was assessed. Spleen cells were cul-

tured in the presence of 0ƒÀG or zymosan and the super-

natants were analyzed for the presence of these cytokines.

Figure 3 shows the dose-dependent effects of 0ƒÀG and

zymosan on the secretion of IL-2, IFN-7 and IL-4. Sim-

ilar kinetics of cytokine secretion were observed by the

stimulation provided by 0ƒÀG or zymosan. However,

0ƒÀG appeared to induce a stronger IL-2 response than

zymosan.

In Vivo Effects of OPG: Cell Kinetics and Differential

Cell Counts in the Peritoneal Cavity following OƒÀG

Administration

To determine whether there was a quantitative change

in leucocyte populations after the in vivo treatment with

0ƒÀG, the number and populations of leucocytes in the

peritoneal cavity were examined in mice treated IP with

0ƒÀG (500 jig), zymosan (500 ƒÊg) or PBS (Fig. 4). This

dose of 0ƒÀG was shown to be optimal for the protection

of immunosuppressed mice against coccidia infection

(50) and therefore was chosen for this study. Mice

injected with PBS were used as a control and showed no

significant changes in leucocyte numbers throughout

the experiment. Both 0ƒÀG and zymosan-treated groups

showed significant increases (PC•ƒ0.05•ƒPC 0.01) in leu-

cocyte numbers at various time points compared to the

control group. The highest infiltration of leucocytes

was observed following zymosan administration.

Zymosan stimulated primarily the infiltration of neu-

trophils and macrophages with the appearance of

increased numbers of lymphocytes 10 days after treat-

ment. The infiltration of cells following 0ƒÀG adminis-

tration consisted almost solely of macrophages, in num-

bers similar to those observed for zymosan.

Intraperitoneal Challenge with S. auretis

To examine whether the administration of 0ƒÀG could

influence the course of S. aureus infection, we assessed

the survival of mice IP challenged with different doses of

S. aureus 3 days after the IP administration of 0ƒÀG

(500ƒÊg), zymosan (500 ƒÊg) or PBS. The results indi-

cated that both 0ƒÀG and zymosan treatments induced a

significant (P<0.05•ƒPC 0.01) protection to the chal-

lenge of 5 X 108 bacteria when compared to the PBS-

treated control group (Table I).

Discussion

Previous studies have shown that ƒÀ-glucans extracted

from yeast or fungi sources potentiate immune respons-

es (2, 19, 20, 24). Our objective in the present study was

to examine the immunomodulatory activities ofƒÀ-glucan

extracted from oats. The results demonstrated that 0ƒÀG

triggers immune functions both in vivo and in vitro.

Fig. 4. Time-dependent effect of the IP injection of oat ƒÀ-glucan

(S), zymosan (•›) or PBS (• ) to mice on the total number of

peritoneal leucocytes, macrophages, neutrophils and lympho-

cytes. Peritoneal fluids were collected at the indicated days and the

number of cells counted and differentiated. Data are expressed as

the mean total cell numbers per 5 ml of peritoneal fluid •} SEM.

Table 1. Protection against intraperitoneal challenge with S. aureus by oat ƒÀ-glucan, zymosan or PBS in mice

99 6 A. ESTRADA ET AL

The effects of 013G were compared to those of zymosan,

a cell wall product of Saccharomyces cerevisiae corn-

posed of carbohydrate polymers such as a-D-mannans

and f3-D-glucans with 1•¨3, 1•¨6 linkages (9) and pre-

sents immunostimulating activities (28).

The anti-microbial activities of ƒÀ-glucans have been

ascribed to their ability to stimulate a wide range of

immunological activities. These activities are important

in host defense mechanisms against infections, including

activation of macrophages, T and NK cells (23, 31, 46)

and the complement system (39). Because the 13-glucans

bind and stimulate cells with multiple immunomodula-

tory activities, the mechanisms of protection provided by

the ƒÀ-glucans are complex.

The ability of 0ƒÀG to stimulate the release of IL-1 is

akin to the in vitro effects of yeast ƒÀ-glucan on

macrophages with the induction of IL-1 in a dose and

time-dependent manner (1, 20). Mouse peritoneal

macrophages have been demonstrated to possess ƒÀ-glu-

can receptors that recognize and mediate phagocytosis of

both glucan and zymosan particles (12). Many of the

immunostimulatory effects attributed to yeast and fungal

β-glucans result from the activation of macrophages (8,

33, 38) in particular, the ability to induce the secretion of

IL-1 (1). IL-1 is an immunoregulatory cytokine with a

wide range of biological activities (10), including induc-

tion of resistance to bacterial infections (7, 22, 42).

Thus, it is possible that IL-1 secreted by OƒÀG-activated

macrophages plays an important role in the resistance to

bacterial challenge observed in the in vivo experiment.

The treatment of peritoneal and cell line macrophages

with OƒÀG induced the release of lesser amounts of TNF-

a in the culture supernatants than zymosan (Fig. 2).

This may represent a desirable effect for the in vivo use

of OƒÀG and explains the apparent low toxicity of OƒÀG to

mice. Lipopolysaccharides, which stimulate the pro-

duction and release of both IL-1 and TNF-a cytokines,

are toxic at low concentrations (44). Yeast 13-glucan

has been shown to induce TNF-a release from

macrophages (1), however, other studies suggest that

β-glucan may also be capable of down-regulating the

expression of TNF-ct in vitro (16) and in vivo (26). The

reduced release of TNF-a from macrophages by ƒÀ-glu-

cans does not appear to result in a generalized inactiva-

tion of the macrophages.

The kinetics of IL-1 and TNF-a production by the

P388D1 macrophage cell line and peritoneal macrophages

in response to 0ƒÀG is time-dependent. Indeed, TNF-a is

detected earlier than IL-1 in both the P388D1 cell line

(48 hr versus 7 days) and peritoneal macrophages (6 hr

versus 24 hr). Stimulated P388D1 cell cultures incubated

for prolonged times, 4 and 7 days, do not demonstrate the

presence of TNF-a (data not shown), possibly due to a

rapid uptake of TNF-a by the macrophages. TNF-a

also rapidly disappeared from peritoneal macrophage

culture supernatants. Similar kinetics for induction of

TNF-a and IL-1 have been reported previously (34).

The present study showed that 0ƒÀG can induce the

secretion of IL-2, IFN-y and IL-4 cytokines from spleen

cell cultures, suggesting the possibility that these

cytokines are also secreted in vivo following stimulation

provided by 0ƒÀG. This result supports the observation

that the administration of 0ƒÀG to immunosuppressed

mice increased the number of IFN-y and IL-4-secreting

cells in the spleen and mesenteric lymph nodes (50).

In addition, yeast ƒÀ-glucan has been shown to increase

the serum and splenic levels of IL-2 in rats (5) and

enhances IL-2 and IFN-y secretion from stimulated

peripheral blood cells (29). Sakurai et al (32) observed

that the induction of IFN-y production by ƒÀ-glucans

represented one of the principal mechanisms of protec-

tion against severe fungal infection and inflammation.

The mechanism by which 0ƒÀG stimulates the production

of IL-2, IFN-y and IL-4 is not known. It is possible that

the activation of T cells to secrete these cytokines may be

mediated indirectly by macrophage-derived cytokines

elicited in response to 0ƒÀG. However, an alternative

mechanism may be through the direct stimulation of

NK cells by 0ƒÀG. NK cells have been shown to possess

receptors for yeast ƒÀ-glucans (1 1, 43) and it is possible

that activation of NK cells by OƒÀG can lead directly to

the secretion of IFN-y and other cytokines.

The in vivo administration of OƒÀG significantly

enhanced the accumulation of macrophages in the peri-

toneal cavity. This was in contrast to the response to

zymosan which was mainly characterized by recruit-

ment of both neutrophils and macrophages (Fig. 4).

The IP inoculation of mice with yeast ƒÀ-glucan has also

been shown to induce a predominant neutrophilic

response in the peritoneal cavity (25).

Previous experiments in this laboratory revealed the

protective effects of OƒÀG by both oral and parenteral

routes in mice infected with Eimeria vermiformis (50).

In this study we demonstrated that pre-treatment of mice

with OƒÀG also confers protection against infection with

S. aureus. These studies demonstrate that the enhance-

ment of resistance to a bacterial challenge induced by

OƒÀG is similar to that reported for yeast and fungal ƒÀ-

glucans (13, 14, 24). The nature of the mechanisms

involved in the protection is unclear, however, it is pos-

sible that activation and recruitment of mononuclear

phagocytes are important factors involved in the enhance-

ment of protection mechanisms.

In conclusion, these studies suggest that immune

functions up-regulated by the administration of OƒÀG,

may play an important role in providing resistance to var-

IMMUNOMODULATORY ACTIVITIES OF OAT P-GLUCAN 99 7

ious infectious diseases. The enhancement of the specific

and non-specific defense mechanisms against infectious

diseases is of enormous importance in both human and

veterinary medicine. Current pharmacologic treatments

for systemic infections may be enhanced when com-

bined with OƒÀG administration.

This study was supported by the Natural Sciences and Engi-

neering Research Council of Canada and the Saskatchewan Agri-

culture Development Fund.

References

1) Abel, G., and Czop, J.K. 1992. Stimulation of human mono-

cyte ƒÀ-glucan receptors by glucan particles induces produc-

tion of TNF-a and IL-1ƒÀ. Int. J. Immunopharmacol. 14:

1363-1373.

2) Artursson, P., Edman, P., and Ericsson, J.L. 1987. Macro-

phage stimulation with some structurally related polysac-

charides. Scand. J. Immunol. 25: 245-254.

3) Benach, J.L., Habicht, G.S., Holbrook, T.W., and Cook,

J.A. 1982. Glucan as an adjuvant for a murine Babesia

microti immunization trial. Infect. Immun. 35: 947-951.

4) Browder, I.W., Williams, D.L., Kitahama, A., and Di Luzio,

N.R. 1984. Modification of post-operative C. albicans sepsis

by glucan immunostimulation. Int. J. Immunopharmacol.

6: 19-26.

5) Czop, J.K., and Austen, K.F. 1985. A ƒÀ-glucan inhibitable

receptor on human monocytes: its identity with the phago-

cytic receptor for particulate activators of the alternative

complement pathway. J. Immunol. 134: 2588-2593.

6) Czop, J.K., and Kay, J. 1991. Isolation and characteriza-

tion of ƒÀ-glucan receptors on human mononuclear phago-

cytes. J. Exp. Med. 173: 1511-1520.

7) Czuprynski, C.J., and Brown, J.F. 1987. Recombinant murine

interleukin-la enhancement of non-specific antibacterial

resistance. Infect. Immun. 55: 2061-2065.

8) Daum, T., and Rohrbach, M.S. 1992. Activation of alveolar

macrophage arachidonic acid metabolism by particulate ƒÀ-

1,3-glucan. FEBS Lett. 309: 119-122.

9) Di Carlo, F.J., and Fiore, J.V. 1958. On the composition of

zymosan. Science 127: 756-757.

10) Dinarello, C.A. 1992. Role of Interleukin-1 in infectious

diseases. Immunol. Rev. 127: 120-146.

11) Duan, X., Ackerly, M., Vivier, E., and Anderson, P. 1994.

Evidence for involvement of ƒÀ-glucan-binding cell surface

lectins in human natural killer cell function. Cell. Immunol.

157: 393-402.

12) Goldman, R. 1988. Characteristics of the ƒÀ-glucan receptor

of murine macrophages. Exp. Cell Res. 174: 481-490.

13) Goldman, R., and Jaffe, C.L. 1991. Administration of ƒÀ-glu-

can following Leishmania major infection suppresses disease

progression in mice. Parasite Immunol. 13: 137-145.

14) Hashimoto, K., Suzuki, I., and Yadomae, T. 1991. Oral

administration of SSG, a ƒÀ-glucan obtained from Sclero-

tinia sclerotiorum, affects the function of Peyer's patches

cells. Int. J. Immunopharmacol. 13: 437-442.

15) Hoffman, O.A., Olson, E.J., and Limper, A.H. 1993. Fungal

β-glucans modulate macrophage release of tumor necrosis

factor-a in response to bacterial lipopolysaccharide. Immunol.

Lett. 37: 19-25.

16) Hoffman, O.A., Standing, i.E., and Limper, A.H. 1993.

Pneuiiiocvstis carinii stimulates tumor necrosis factor-a

release from alveolar macrophages through a f3-glucan-

mediated mechanism. J. Immunol. 150: 3932-3940.

17) Holbrook, T.W., Cook, J.A., and Parker, B.W. 1981. Immu-

nization against Leishmania donovani: glucan as an adjuvant

with killed promastigotes. Am. J. Trop. Med. Hyg. 30: 762-

768.

18) Jeraci, J.L., and Lewis, B.A. 1989. Determination of soluble

fiber components: (1•¨3, 1•¨4)-ƒÀ-D-glucans and pectins.

Anim. Feed Sci. Technol. 23: 15-25.

19) Konopski, Z., Rasmussen, L.T., Seljelid, R., and Eskeland, T.

1991. Phagocytosis of ƒÀ-1,3-D-glucan-derivatized microbeads

by mouse peritoneal macrophages involves three different

receptors. Scand. J. Immunol. 33: 297-306.

20) Konopski, Z., Seljelid, R., and Eskeland, T. 1993. Cytokines

and PGE, modulate the phagocytic function of the ƒÀ-glucan

receptor in macrophages. Scand. J. Immunol. 37: 587-592.

21) Manners, D.J., Masson, A.J., and Patterson, J.C. 1973. The

structure of a ƒÀ-(1•¨)-D-glucan from yeast cell walls.

Biochem. J. 135: 19-30.

22) McIntyre, K.W., Unowsky, J., DeLorenzo, W., and Ben-

jamin, W. 1989. Enhancement of antibacterial resistance of

neutropenic, bone marrow-suppressed mice by interleukin-

Ia. Infect. Immun. 57: 48-54.

23) Okazaki, M., Adachi, Y., Ohno, N., and Yadomae, T. 1995.

Structure-activity relationship of (1•¨3)-ƒÀ-D-glucans in the

induction of cytokine production from macrophages in vitro.

Biol. Pharm. Bull. 18: 1320-1327.

24) Onderdonk, A.B., Cisneros, R.L., Hinkson, P., and Ostroff,

G. 1992. Anti-infective effect of poly-ƒÀ1-6-glucotriosyl-

β1-3-glucopyranose glucan in vivo. Infect. Immun. 60:1642-1647.

25) Rasmussen, L.T., and Seljelid, R. 1990. Dynamics of blood

components and peritoneal fluid during treatment of murine

E. coli sepsis with ƒÀ-1,3-D-polyglucose derivatives. Scand. J.

Immunol. 32: 321-331.

26) Rasmussen, L.T., Konopski, Z., Oian, P., and Seljelid, R.

1992. Killing of Escherichia coli by mononuclear phagocytes

and neutrophils stimulated in vitro with ƒÀ-1,3-D-polyglu-

cose derivatives. Microbiol. Immunol. 36: 1173-1188.

27) Reynolds, J.A., Kastello, M.D., Harrington, D.G., Crabbs,

C.L., Peters, C.J., Jemski, J.V., Scott, G.H., and Di Luzio,

N.R. 1980. Glucan-induced enhancement of host resistance

to selected infectious diseases. Infect. Immun. 30: 51-57.

28) Riggi, S.J., and Di Luzio, N.R. 1961. Identification of a

reticuloendothelial stimulating agent in zymosan. Am. J.

Pathol. 200: 297-300.

29) Sakagami, Y., Mizoguchi, Y., Shin, T., Seki, S., Kobayashi,

K., Morisawa, S., and Yamamoto, S. 1988. Effects of an anti-

tumor polysaccharide, schizophyllan on interferon-ƒÁ and

interleukin-2 production by peripheral blood mononuclear

cells. Biochem. Biophys. Res. Commun. 155: 650-657.

30) Sakurai, T., Hashimoto, K., Suzuki, I., Ohno, N., Oikawa, S.,

Masuda, A., and Yadomae, T. 1992. Enhancement of murine

alveolar macrophage functions by orally administered p-

99 8 A. ESTRADA ET AL

glucan. Int. J. Immunopharmacol. 14: 821-830.

31) Sakurai, T., Ohno, N., and Yadomae, T. 1994. Changes in

immune mediators in mouse lung produced by administration

of soluble (1•¨3)-ƒÀ-D-glucan. Biol. Pharm. Bull. 17: 617-

622.

32) Sakurai, T., Ohno, N., and Yadomae, T. 1996. Effects of

fungal ƒÀ-glucan and IFN-ƒÁ on the secretory functions of

murine alveolar macrophages. J. Leukocyte Biol. 60: 118-

124.

33) Seljelid, R., Rasmussen, L.T., Larm, O., and Hoffman, J.

1987. The protective effect of ƒÀ- 1-3-D-glucan-derivatized

plastic beads against Escherichia coli infection in mice.

Scand. J. Immunol. 25: 55-60.

34) Seljelid, R., Figenschau, Y., Bogwald, J., Rasmussen, L.T.,

and Austgulen, R. 1989. Evidence that tumor necrosis by

aminated polyglucose is mediated by a concerted

action of local and systemic cytokines. Scand. J. Immunol.

30: 687-694.

35) Soltys, J., Benkova, M., and Boroskova, Z. 1994. Im-

munorestorative effect of glucan immunomodulator on

guinea pigs with experimental ascariosis. Vet. Immunol.

Immunopathol. 42: 379-388.

36) Suga, T., Shiio, T., Maeda, Y., and Chihara, G. 1984. Anti-

tumor activity of lentinan in murine syngeneic and autochtho-

nous hosts and its suppressive effect on 3-methylcholan-

threne-induced carcinogenesis. Cancer Res. 44: 5132-5137.

37) Suzuki, I., Hashimoto, K., Ohno, N., Tanaka, H., and Yado-

mae, T. 1989. Immunomodulation by orally administered 3-

glucan in mice. Int. J. Immunopharmacol. 11: 761-769.

38) Suzuki, I., Tanaka, H., Kinoshita, A., Oikawa, S., Osawa, M.,

and Yadomae, T. 1990. Effect of orally administered ƒÀ-glu-

can on macrophage function in mice. Int. J. Immunophar-

macol. 12: 675-684.

39) Thornton, B.P., Vetvicka, V., Pitman, M., Goldman, R.C., and

Ross, G.D. 1996. Analysis of the sugar specificity and mo-

lecular location of the ƒÀ-glucan-binding lectin site of com-

plement receptor type 3 (CD1 lb/CD18). J. Immunol. 156:

1235-1246.

40) Tvaroska, I., Ogawa, K., Deslandes, Y., and Marchessault,

R.H. 1983. Crystalline conformation and structure of lichenan

and barley ƒÀ-glucan. Can. J. Chem. 61: 1608-1616.

41) Vacha, J., Znojil, V., Pospisil, M., Hola, J., and Pipalova, I.

1994. Microcytic anemia and changes in ferrokinetics as

late after-effects of glucan administration in murine hepati-tis virus-infected C57BL/10ScSnPh mice. Int. J. Immunophar-macol. 16: 51-60.

42) Van der Meer, J.W.M., Barza, M., Wolff, S.M., and Dinarel-lo, C.A. 1988. Low dose recombinant interleukin-1 pro-tects granulocytopenic mice from lethal Gram-negative

infection. Proc. Natl. Acad. Sci. U.S.A. $5: 1620-1623. 43) Vetvicka, V., Thornton, BR, and Ross, G.D. 1996. Soluble

β-glucan polysaccharide binding to the lectin site of neu-

trophil or natural killer cell complement receptor type 3

(CD11b/CD18) generates a primed state of the receptor

capable of mediating cytotoxicity of iC3b-opsonized target

cells. J. Clin. Invest. 98: 50-61.

44) Waage, A. 1987. Production and clearance of tumor necro-

sis factor in rats exposed to endotoxin and dexamethasone.

Clin. Immunol. Immunopathol. 45: 348-353.

45) Williams, D.L., and Di Luzio, N.R. 1980. Glucan-induced

modification of murine viral hepatitis. Science 208: 67-69.

46) Williams, D.L., Sherwood, E.R., McNamee, R.B., Jones,

E.L., and Di Luzio, N.R. 1985. Therapeutic efficacy of glu-

can in a murine model of hepatic metastatic disease. Hepa-

tology 5: 198-206.

47) Williams, D.L., Pretus, H.A., McNamee, R.B., Jones, E.L.,

Ensley, H.E., Browder, I.W., and Di Luzio, N.R. 1991.

Development, physicochemical characterization and pre-

clinical efficacy evaluation of a water soluble glucan sulfate

derived from Saccharomyces cerevisiae. Immunopharma-

cology 22: 139-155.

48) Wood, P.J., Weisz, J., and Blackwell, B.A. 1991. Molecular

characterization of cereal ƒÀ-D-glucans. Structural analysis of

oat ƒÀ-D-glucan and rapid structural evaluation of ƒÀ-D-glucans

from different sources by high-performance liquid chro-

matography of oligosaccharides released by lichenase. Cere-

al Chem. 68: 31-39.

49) Wood, P.J., Weisz, J., and Fedec, P. 1991. Potential for ƒÀ-glu-

can enrichment in brans derived from oat (Avena saliva L.)

cultivars of different (1•¨3), (1l•¨4)-ƒÀ-D-glucan concentra-

tions. Cereal Chem. 68: 48-51.

50) Yun, C.H., Estrada, A., Van Kessel, A., Gajadhar, A.A.,

Redmond, M.J., and Laarveld, B. 1997. ƒÀ-( 1•¨3,1•¨4) Oat

glucan enhances resistance to Eimeria vermiformis infection

in immunosuppressed mice. Int. J. Parasitol. 27: 329-337.