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Infection and Immunity, January 1999, p. 244-252, Vol. 67, No. 1
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Modulation of Endotoxin- and Enterotoxin-Induced Cytokine Release
by In Vivo Treatment with
-(1,6)-Branched
-(1,3)-Glucan
Jindrich
Soltys, and
Mark T.
Quinn*
Department of Veterinary Molecular Biology,
Montana State University, Bozeman, Montana 59717
Received 8 May 1998/Returned for modification 17 July 1998/Accepted 20 August 1998
 |
ABSTRACT |
Leukocytes activated by endotoxin or enterotoxins release
proinflammatory cytokines, thereby contributing to the cascade of events leading to septic shock. In the present studies, we analyzed the
effects of in vivo administration of a soluble immunomodulator,
-(1,6)-branched
-(1,3)-glucan (soluble
-glucan), on
toxin-stimulated cytokine production in monocytes and lymphocytes
isolated from treated mice. In vitro stimulation of lymphocytes
isolated from soluble
-glucan-treated mice with lipopolysaccharide
(LPS) resulted in enhanced production of interleukin-6 (IL-6) and
suppressed production of tumor necrosis factor alpha (TNF-
), while
stimulation of these cells with staphylococcal enterotoxin B (SEB) or
toxic shock syndrome toxin 1 (TSST-1) resulted in enhanced production of gamma interferon (IFN-
) and suppressed production of IL-2 and
TNF-
compared to that in cells isolated from untreated mice. In
vitro stimulation of monocytes isolated from soluble
-glucan-treated mice with LPS also resulted in suppressed TNF-
production, while stimulation of these cells with SEB or TSST-1 resulted in suppressed IL-6 and TNF-
production compared to that in cells isolated from untreated mice. Thus, the overall cytokine pattern of leukocytes from
soluble
-glucan-treated mice reflects suppressed production of
proinflammatory cytokines, especially TNF-
. Taken together, our
results suggest that treatment with soluble
-glucan can modulate the
induction cytokines during sepsis, resulting in an overall decrease in
host mortality.
 |
INTRODUCTION |
Sepsis caused by gram-negative
bacilli or gram-positive cocci represents a major source of morbidity
and mortality in medical facilities today (17, 34, 53, 71).
The reasons for the high incidence of bacterial sepsis are probably
related to several key factors. First, increased bacterial virulence
and drug resistance have complicated treatment and led to problems in
tracking disease-causing pathogens (50, 69). Second, the
host defense capacity of many patients has been compromised by the
increased use of immunosuppressive therapies (33, 35, 38).
Finally, the incidence of opportunistic infections has grown rapidly
due to the worldwide AIDS epidemic (33, 38, 60). To combat
the problems of drug resistance, a significant amount of research has
focused on the development of antibiotics that counteract resistance
(50, 69). However, it will be some time before these agents
become available, and, as has occurred in the past, the introduction of
these agents may eventually lead to the emergence of resistant
pathogens (29, 50). Thus, there is clearly a need to develop
and characterize alternative anti-infective substances as
adjuvants to classical antibiotic therapies.
One of the most promising recent alternatives to classical antibiotic
treatment is the use of immunomodulators for enhancing host defense
responses (5-7). Several types of immunomodulators have
been identified, including mammalian proteins such as gamma interferon
(IFN-
) (22, 48), granulocyte colony-stimulating factor
(49), and granulocyte-macrophage colony-stimulating factor (4, 45, 49), as well as substances isolated and purified from microorganisms (12, 14, 31, 37, 59). The latter type of
immunomodulators typically induces nonspecific stimulation of the
immune system. For example,
-(1,3)-glucans purified from fungi and
yeast have been shown to have broad anti-infective activities (reviewed
in references 11 and 80). These
polysaccharide compounds have been shown to bind to receptors on
leukocytes and stimulate a number of immune responses, such as cytokine
release (1, 19, 57), generation of reactive oxygen species
(27, 62), generation of nitric oxide (61), and
release of arachidonic acid metabolites (15, 16, 19, 56).
However, because of the poor solubility and direct
leukocyte-activating action of
-(1,3)-glucans, these
compounds have limited clinical usefulness.
Recently, several soluble derivatives of
-(1,3)-glucan that show
potent immunomodulatory activity have been developed. Aminated
-(1,3)-glucan has been shown to induce resistance to bacterial infection (67) and cause regression of solid tumors in mice (66). In addition, a combination of IFN-
with aminated
-(1,3)-glucan resulted in better inhibition of the growth of mouse
liver metastases than did either of these agents alone (73).
Soluble
-(1,6)-branched
-(1,3)-glucan (soluble
-glucan) has
also been shown to enhance microbicidal activities of neutrophils and
macrophages but, in contrast to aminated
-(1,3)-glucan
(19), has been reported to have no direct activating effect
on neutrophil or monocyte functions (11, 55). Rather,
soluble
-glucan appears to prime leukocytes for an enhanced host
defense response when they are exposed to a secondary stimulus, such as
phorbol myristate acetate, N-formylated chemotactic peptide, or
opsonized bacteria (11, 13, 51, 76, 79). Thus, soluble
-glucan represents a potentially useful immunomodulator, and it has
been shown to reduce postoperative infection rates and reduce the
length of hospitalization in clinical trials (5, 6).
Although glucan-derived substances have been extensively studied as
immunomodulators for treatment of a number of bacterial, fungal,
parasitic, and viral infections (reviewed in reference 11), less is known about how these substances exert
their biological effects. Aminated
-(1,3)-glucan has been shown to
stimulate the production of interleukin-1 (IL-1), tumor necrosis factor
alpha (TNF-
), and prostaglandin E2 by human monocytes
(19) and murine macrophages (68). Soluble
-glucan has been shown to prime the oxidative burst and microbicidal
activity of human neutrophils (42, 77) and appears to bind
to a unique
-glucan receptor that recognizes soluble
-glucan in
these cells (77, 78). Soluble
-glucan has also been shown
to increase total leukocyte numbers, enhance the clearance of
bacteria from the blood, and reduce mortality in rat sepsis models
(13, 51, 76). In more recent studies, Liang et al.
(40) showed that soluble
-glucan enhanced the
clearance of a multidrug-resistant strain of Staphylococcus aureus in a rat intra-abdominal sepsis model and found that this effect was accompanied by increased leukocyte numbers in blood and
enhancement of the oxidative burst.
Because of the potential usefulness of
-(1,3)-glucan derivatives as
immunomodulators, it is of interest to further characterize the
mechanism(s) whereby soluble
-glucan modulates the host defense response. Therefore, we analyzed the effects of in vivo administration of soluble
-glucan on in vitro cytokine production by endotoxin- and
enterotoxin-stimulated lymphocytes and monocytes. Production of the
most important cytokines involved in the pathogenesis of septic shock
(IL-2, IL-6, IFN-
, and TNF-
) in cells isolated from untreated and
soluble
-glucan-treated mice was analyzed. In addition, the level of
apoptosis in lymphocytes and monocytes isolated from these mice was
examined. Our results show that toxin-stimulated cytokine release and
leukocyte apoptosis can be modulated by in vivo administration of
soluble
-glucan in a mouse model.
 |
MATERIALS AND METHODS |
Animals.
Pathogen-free female BALB/cby inbred mice, aged 6 to 8 weeks, were obtained from the Animal Resource Center, Montana
State University. All the mice were housed in accordance with approved guidelines and were provided with food and water ad libitum. All animal
use was approved by the Montana State University Animal Care and Use Committee.
Drug treatment.
For each experiment, soluble
-glucan
(Alpha-Beta Technology, Worcester, Mass.) was administered
intramuscularly to 10 mice at a dose of 1 mg/kg of body weight. An
equal number of mice served as controls and received no injection. The
cells from each group of mice were then isolated, pooled, and analyzed
as described below. Three independent experiments were performed to
obtain the data presented.
Leukocyte isolation.
Spleen lymphocytes and bone marrow
monocytes from both experimental groups were isolated aseptically
24 h after in vivo soluble
-glucan administration. To isolate
spleen lymphocytes, the spleens were stripped of fat and cut into small
pieces. Single-cell suspensions were made by pressing spleen pieces
through 70-µm-mesh cell strainers (Becton Dickinson) into RPMI 1640 (Cellgro). After removal of erythrocytes by hypotonic lysis, the cell
suspension was depleted of macrophages and monocytes by adherence to
plastic tissue culture dishes for 40 min at 37°C. Purified cell
suspensions were washed three times in complete RPMI 1640 supplemented
with 10% fetal calf serum and used for in vitro cytokine and apoptosis
experiments. Analysis of the isolated cells by light microscopy and by
flow cytometry showed that >95% of the cells were lymphocytes.
Bone marrow monocytes were harvested by washing mouse femurs with
Dulbecco's phosphate-buffered saline by standard methods (21). The cells were then centrifuged and resuspended in
RPMI 1640. The mononuclear cells were separated by density gradient centrifugation on Histopaque gradients (18, 21). Purified cell suspensions were washed three times in complete RPMI 1640 supplemented with 10% fetal calf serum and used for in vitro cytokine and apoptosis experiments. Analysis of the isolated cells by flow cytometry with lineage-specific antibodies showed that >93% of the
cells were monocytes while the remaining cells were lymphocytes.
The viability of the cells used throughout was

95%. Note that the
reagents and labware used in all experiments were lipopolysaccharide
(LPS)
free.
Measurement of cytokine production in vitro.
Aseptically
isolated lymphocytes and monocytes were diluted at 5 × 106 cells/ml in complete RPMI 1640 and incubated separately
with 10 µg of soluble
-glucan per ml, Toxic shock syndrome toxin 1 (TSST-1) (10 µg/ml; Toxin Technologies, Sarasota, Fla.),
staphylococcal enterotoxin B (SEB) (10 µg/ml; Toxin Technologies), or
LPS from E. coli K-235 (10 µg/ml; Sigma Chemical Co., St.
Louis, Mo.) for the indicated times at 37°C under 5%
CO2. After incubation, the cells were removed by
centrifugation and the collected supernatants were stored at
20°C
until analyzed.
Cytokine levels in culture supernatants were determined by a standard
sandwich enzyme-linked immunosorbent assay technique
(
2).
Briefly, Nunc MaxiSorp (Nalge Nunc International, Roskilde,
Denmark)
plates were coated for 12 h at 4°C with rat anti-murine
IL-2,
IL-6, IFN-

, or TNF-

monoclonal antibodies (PharMingen,
San Diego,
Calif.), the plates were blocked, supernatant samples
were added, and
the plates were incubated overnight at 4°C. The
plates were then
washed and incubated for 90 min at room temperature
with
biotin-conjugated rat anti-murine IL-2, IL-6, TNF-

, or IFN-
monoclonal antibodies followed by an alkaline phosphatase-conjugated
goat anti-biotin monoclonal antibody (Vector Laboratories, Inc.,
Burlingame, Calif.). The fluorescent substrate for alkaline
phosphatase,
4-methylumbelliferyl phosphate dicyclohexylammonium salt
(Molecular
Probes, Eugene, Oreg.), was used to develop the assay, and
fluorescence
was measured with a Bio-Tek Instruments FL 500 microtiter
plate
reader, using excitation and emission wavelengths of 360 and 460
nm, respectively. To quantify the amount of cytokine present in
test
samples, values were extrapolated from standard curves established
by
analyzing different dilutions of recombinant murine IL-2, IL-6,
IFN-

, and TNF-

. The values shown represent the mean ± standard
error of the mean (SEM) of three independent experiments
(triplicate
samples in each experiment). The detection limits for IL-2,
IL-6,
IFN-

, and TNF-

were 200, 200, 200, and 30 pg/ml,
respectively.
Detection of apoptotic cells.
Apoptosis was analyzed with an
APO DIRECT kit (Pharmingen), which is based on a modification of the
TUNEL method (28, 30). Briefly, lymphocyte and monocyte
samples, diluted at 106 cells/100 µl, were stained for
fragmentation of the genomic DNA by terminal
deoxynucleotidyltransferase-mediated nick end labeling (TUNEL) with
fluorescein isothiocyanate-conjugated dUTP, and the cells were analyzed
by flow cytometry. All samples were analyzed in duplicate.
Statistical analysis.
Unless otherwise indicated, the
results are expressed as the mean ± SEM of data obtained from
triplicate experiments. Statistical analysis was performed by a paired
Student t test. Differences at P < 0.05
were considered statistically significant.
 |
RESULTS |
Endotoxin-induced cytokine release.
To investigate the effects
of in vivo soluble
-glucan treatment on lymphocyte cytokine
production in response to endotoxin or soluble
-glucan in vitro, we
treated mice with soluble
-glucan and isolated spleen lymphocytes as
described in Materials and Methods. As shown in Fig.
1, lymphocytes isolated from untreated and soluble
-glucan-treated mice exhibited some significant
differences in cytokine production.

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FIG. 1.
Endotoxin-stimulated cytokine production by lymphocytes
isolated from untreated and soluble -glucan-treated mice. Spleen
lymphocytes were stimulated in vitro for the indicated times with 10 µg of soluble -glucan (solid bars) or LPS (hatched bars) per ml.
Control cells (open bars) were incubated in culture medium alone. The
cytokine levels in the culture supernatants were measured by
enzyme-linked immunosorbent assay with paired cytokine-specific
antibodies, and the amounts of cytokine are expressed in nanograms or
picograms per milliliter, standardized against mouse recombinant
cytokines. The results represent the mean ± SEM of three
independent experiments. *, statistically significant values
(P < 0.05) compared to the control. , statistically
significant values (P < 0.05) compared to cells
isolated from untreated mice.
|
|
Primary in vitro stimulation of lymphocytes with soluble

-glucan or
LPS resulted in a modest but significantly higher release
of IL-2 at
15 h compared with that from controls, whereas lymphocytes
isolated from soluble

-glucan-treated mice produced increased
levels
of IL-2 only after soluble

-glucan stimulation but not
after LPS
stimulation. At 24 and 48 h, spleen lymphocytes from
both
untreated and soluble

-glucan-treated mice produced significantly
higher levels of IL-2 only in response to in vitro stimulation
by
soluble

-glucan. Overall, the pattern of IL-2 production was
similar
for cells isolated from untreated and soluble

-glucan-treated
mice.
The levels of IL-6 production by soluble

-glucan- or LPS-stimulated
lymphocytes isolated from untreated mice were not significantly
greater
than those in control cells (Fig.
1). However, in lymphocytes
isolated
from soluble

-glucan-treated mice, LPS stimulation in
vitro resulted
in a large increase in IL-6 production in cell
supernatants from 24- and 48-h
incubations.
In lymphocytes isolated from untreated and soluble

-glucan-treated
mice, soluble

-glucan and LPS both stimulated an increase
in IFN-

production at 24 h (Fig.
1). At 48 h, soluble

-glucan
stimulated even higher levels of IFN-

production while the levels
of
IFN-

produced by LPS-stimulated cells had decreased to baseline.
Overall, the pattern of IFN-

production in cells isolated from
soluble

-glucan-treated mice was similar to that observed in
cells
from untreated
mice.
Lymphocytes isolated from untreated and soluble

-glucan-treated mice
produced significant levels of TNF-

at 4 and 8 h in
response to
stimulation with either soluble

-glucan or LPS, although
the
response to LPS was, in general, higher than that to soluble

-glucan
(Fig.
1). However, at 24 h, TNF-

production by cells
from
untreated mice stimulated with either agent had returned
to background
levels. Interestingly, in cells isolated from soluble

-glucan-treated mice, the peak level of stimulated TNF-

production
at 8 h was significantly lower than in comparable
samples from
untreated mice (Fig.
1). In addition, cells from soluble

-glucan-treated
mice were still producing a significant amount of
TNF-

at 24
h of LPS stimulation while lymphocytes from
untreated mice produced
only minimal levels of TNF-

at 24 h.
Thus, in vivo treatment
with soluble

-glucan not only suppresses the
magnitude of lymphocyte
TNF-

production but also may act to prolong
the production of
low levels of this
cytokine.
We also analyzed the effects of in vivo soluble

-glucan treatment on
monocyte cytokine profiles in response to in vitro stimulation.
As
shown in Fig.
2, monocytes isolated from
soluble

-glucan-treated
mice produced increased levels of IL-2 after
soluble

-glucan
or LPS stimulation for 15 and 24 h, compared to
cells from untreated
mice. However, the levels of IL-2 measured here
were very low
(10 times lower than those produced by spleen
lymphocytes) and
may represent IL-2 produced by residual T cells
(normally 5 to
10% of the cells) in our monocyte preparation.

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FIG. 2.
Endotoxin-stimulated cytokine production by monocytes
isolated from untreated and soluble -glucan-treated mice. Bone
marrow monocytes were stimulated in vitro for the indicated times with
10 µg of soluble -glucan (solid bars) or LPS (hatched bars) per
ml. Control cells (open bars) were incubated in culture medium alone.
The cytokine levels in the culture supernatants were measured by ELISA
with paired cytokine-specific antibodies, and the amounts of cytokine
are expressed in nanograms or picograms per milliliter standardized
against mouse recombinant cytokines. The results represent the
mean ± SEM of three independent experiments. *, statistically
significant values (P < 0.05) compared to the control;
, statistically significant values (P < 0.05)
compared to cells isolated from untreated mice.
|
|
Monocytes isolated from untreated mice produced increased levels of
IL-6 when stimulated in vitro with soluble

-glucan or
LPS, although
the response to LPS was three- to fourfold higher.
In contrast,
monocytes isolated from soluble

-glucan-treated
mice produced high
levels of IL-6 after in vitro stimulation with
LPS but not after
stimulation with soluble

-glucan (Fig.
2).
Overall, the pattern of
IL-6 production in cells isolated from
soluble

-glucan-treated mice
was similar to that observed in
cells from untreated
mice.
As shown in Fig.
2, monocytes isolated from both untreated and soluble

-glucan-treated mice produced significant levels of
TNF-

after 4, 8, and 24 h of stimulation with LPS, although the
levels produced
by cells from soluble

-glucan-treated mice were
significantly lower
than in comparable cells from untreated mice.
Soluble

-glucan
stimulation of monocytes from untreated mice
for 8 and 24 h
resulted in the production of low levels of TNF-

,
whereas no TNF-

was produced in response to in vitro stimulation
by soluble

-glucan.
Consistent with our lymphocyte analyses (Fig.
1), these results show
that in vivo treatment with soluble

-glucan
also suppresses the
magnitude of endotoxin-stimulated TNF-

production
in
monocytes.
Enterotoxin-induced cytokine release.
Enterotoxins (also known
as superantigens) from gram-positive bacteria play an important role in
septic shock by activating T cells, resulting in the release of
inflammatory cytokines (46, 47, 72). Therefore, we also
analyzed the effects of in vivo soluble
-glucan treatment on
lymphocyte cytokine profiles in response to enterotoxins, such as SEB
and TSST-1. As shown in Fig. 3,
lymphocytes isolated from untreated and soluble
-glucan treated mice
also exhibited some important differences in cytokine production, and
the most significant changes were associated with IL-2, IFN-
, and
TNF-
. Lymphocytes isolated from untreated mice released significant
amounts of IL-2 after stimulation with SEB or TSST-1 (Fig. 3). In
contrast, in vitro stimulation of cells isolated from soluble
-glucan-treated mice with SEB or TSST-1 resulted in a modest but
significantly decreased production of IL-2 (Fig. 3).

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FIG. 3.
Enterotoxin-stimulated cytokine production by
lymphocytes isolated from untreated and soluble -glucan-treated
mice. Spleen lymphocytes were stimulated in vitro for the indicated
times with 10 µg of SEB (solid bars) or TSST-1 (hatched bars) per ml.
Control cells (open bars) were incubated in culture medium alone. The
cytokine levels in the culture supernatants were measured by ELISA with
paired cytokine-specific antibodies, and the amounts of cytokine are
expressed in nanograms or picograms per milliliter, standardized
against mouse recombinant cytokines. The results represent the
mean ± SEM of three independent experiments. *, statistically
significant values (P < 0.05) compared to the control;
, statistically significant values (P < 0.05)
compared to cells isolated from untreated mice.
|
|
In lymphocytes isolated from untreated and soluble

-glucan-treated
mice, SEB and TSST-1 both stimulated IL-6 production at
15, 24, and
48 h. Although the responses to SEB and TSST-1 were
significantly
different at 24 and 48 h in cells isolated from
the treated mice
(Fig.
3), the overall pattern of IL-6 production
was fairly similar
between cells isolated from soluble

-glucan-treated
and untreated
mice.
Lymphocytes isolated from untreated mice also produced significant
levels of IFN-

when stimulated with SEB or TSST-1. In
contrast to
the IL-2 response, however, cells from soluble

-glucan-treated
mice
exhibited enhanced IFN-

production at 15 and 24 h compared
to
the production after similar incubation times for cells from
untreated
mice (Fig.
3). At 48 h, similar levels of IFN-

were
produced by
cells from both treated and untreated
mice.
Lymphocytes isolated from untreated and soluble

-glucan-treated mice
produced significant levels of TNF-

at 4, 8, and 24
h (with a
peak at 8 h) in response to stimulation with either
SEB or TSST-1,
although the responses were significantly lower
in cells from soluble

-glucan-treated mice than in cells from
untreated mice (Fig.
3).
Again, these results demonstrate that
in vivo treatment with soluble

-glucan suppresses the magnitude
of toxin-stimulated lymphocyte
TNF-
production.
We also analyzed the effects of in vivo soluble

-glucan treatment on
monocyte cytokine profiles in response to enterotoxin.
As shown in Fig.
4, monocytes isolated from untreated mice
produced
higher levels of IL-2 than did control cells at 15, 24, and
48
h after stimulation with SEB or TSST-1. In contrast, the
response
of monocytes isolated from soluble

-glucan-treated mice was
significantly
lower at 15 and 24 h (no significant increase over
controls) and
significant levels of IL-2 were observed only after
48 h of incubation
with the toxins. Again, the levels of IL-2
measured here were
very low and may represent IL-2 produced by residual
T cells in
our monocyte preparation.

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FIG. 4.
Enterotoxin-stimulated cytokine production by monocytes
isolated from untreated and soluble -glucan-treated mice. Bone
marrow monocytes were stimulated in vitro for the indicated times with
10 µg of SEB (solid bars) or TSST-1 (hatched bars) per ml. Control
cells (open bars) were incubated in culture medium alone. The cytokine
levels in the culture supernatants were measured by ELISA with paired
cytokine-specific antibodies, and the amounts of cytokine are expressed
in nanograms or picograms per milliliter, standardized against mouse
recombinant cytokines. The results represent the mean ± SEM of
three independent experiments. *, statistically significant values
(P < 0.05) compared to the control; , statistically
significant values (P < 0.05) compared to cells
isolated from untreated mice.
|
|
Both monocytes from untreated and soluble

-glucan-treated mice
produced IL-6 after stimulation with SEB; however, the responses
were,
in general, significantly lower in cells isolated from the
treated mice
(Fig.
4).
As shown in Fig.
4, monocytes isolated from both untreated and soluble

-glucan-treated mice produced significant levels of
TNF-

after
stimulation with SEB or TSST-1, although the levels
produced by cells
isolated from soluble

-glucan-treated mice
were significantly lower
at all time points than were those produced
by comparable cells from
untreated mice. Again, these results
show that in vivo treatment with
soluble

-glucan suppresses the
magnitude of stimulated TNF-

production in
monocytes.
Apoptosis.
Spleen lymphocytes isolated from untreated mice and
stimulated in vitro with LPS, SEB, TSST-1, or soluble
-glucan showed slight increases in the percentage of apoptotic cells at 12 h (Fig. 5) and 24 h (results not
shown) compared to control unstimulated cell populations. In general,
lymphocytes isolated from soluble
-glucan-treated mice showed a
higher level of apoptosis than did cells isolated from untreated mice,
especially in control cell populations (Fig. 5). One key exception was
in response to LPS, where a significantly lower level of apoptosis was
observed in cells isolated from soluble
-glucan-treated mice. We
observed a similar pattern of responses after 24 h of incubation,
although the overall level of apoptosis was increased (data not shown).

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FIG. 5.
Analysis of monocyte and lymphocyte apoptosis in cells
isolated from untreated and soluble -glucan-treated mice.
Toxin-stimulated apoptosis was analyzed in lymphocytes and monocytes
from untreated (open bars) and soluble -glucan-treated (hatched
bars) mice at 12 h after isolation. The results are expressed as
the percentage of total cells and represent the mean ± SEM of two
independent experiments. *, statistically significant values
(P < 0.05) compared to cells isolated from untreated
mice.
|
|
Analysis of monocytes isolated from untreated mice showed a slightly
lower overall level of apoptosis at 12 h (Fig.
5) and
24 h
(results not shown) compared to lymphocytes isolated from
the same
mice, and none of the agents tested induced an increase
in the level of
apoptosis (Fig.
5). In contrast, LPS and TSST-1
were able to induce
increased levels of apoptosis in monocytes
isolated from soluble

-glucan-treated mice (Fig.
5). Again, we
observed a similar pattern
of responses after 24 h of incubation,
although the overall level
of apoptosis was increased (data not
shown).
 |
DISCUSSION |
The mechanism(s) of modulation of the immune response by
immunomodulators, such as soluble
-glucan, is not well understood. In previous studies, soluble
-glucan has been shown to enhance host
defense mechanisms in animal infection models (13, 39, 40,
76) as well as in humans (5, 6). Considering the key
role that leukocyte cytokine production plays in the pathogenesis of
septic shock (76), we have examined how in vivo
administration of soluble
-glucan could modulate in vitro primary
and secondary cytokine responses in endotoxin- and enterotoxin-treated cells.
Primary stimulation of lymphocytes and monocytes isolated from
untreated mice with LPS, SEB, or TSST-1 resulted in cytokine profiles
characteristic for the induction of septic shock (24, 34,
47); i.e., these cells released high levels of all of the most
important proinflammatory cytokines. In contrast, lymphocytes and
monocytes isolated from mice treated in vivo with soluble
-glucan
produced a significantly different cytokine response, which was
generally characterized by enhanced IFN-
production and suppressed
production of TNF-
, suggesting that immunomodulation with soluble
-glucan might act to depress the inflammatory cytokine response.
Interestingly, we observed that soluble
-glucan itself stimulated
modest cytokine production by leukocytes isolated from untreated and
soluble
-glucan-treated mice, while others have reported previously
that some forms of soluble
-glucan did not induce cytokine
production in vitro by human leukocytes (55, 77) or cultured
murine BMC2.3 cells (3). In addition, Liang et al.
(40) reported that soluble
-glucan did not induce the production of IL-1
or TNF-
in vivo in soluble
-glucan-treated rats. Possible explanations for these differences are related to the
type of cells analyzed or differences in assay sensitivity; however,
further studies are necessary to investigate this issue.
Currently, several experimental approaches are used in the treatment of
bacterial sepsis (53). For example, antibodies against CD14,
LPS-binding protein, or LPS itself have been used for the treatment of
endotoxemia (41, 44, 75). Other reported treatments involve
the use of soluble receptors (23) or monoclonal antibodies (25, 54, 63) against TNF-
for treatment of septic shock. In the present studies, we show that in vivo administration of soluble
-glucan suppressed TNF-
production and elevated IFN-
production; these changes may play an important protective role in the
outcome of septic shock. This idea is further supported by studies by
Barton and Jackson (9), who found that pretreatment with an
antibody to TNF-
protected mice from LPS-induced septic shock and
that mortality was reduced even more by treatment with a combination of
recombinant IL-6 and a low dose of the anti-TNF-
antibody. Thus,
based on our present studies, we conclude that stimulation of the
reticuloendothelial system by soluble
-glucans (11) acts,
in part, to modulate the production of proinflammatory and
anti-inflammatory cytokines during sepsis, resulting in an overall
decrease in host mortality (13, 76).
Regulation of apoptosis has also been proposed to play an important
role in the modulation of the inflammatory response (64, 65,
70). It appears, however, that the level of apoptosis is
regulated by a complex interplay of cytokines encountered by the cell
(43, 81). For example, the major outcome of exposure to
endotoxin or enterotoxins is the production of a cascade of proinflammatory cytokines (24, 47), where TNF-
can
regulate the production of IL-1
and IL-6 (10, 26), and
both TNF-
and IL-6 play key roles in the modulation of apoptosis
(81). Cytokines not only modulate apoptosis but also can
enhance the capacity of the macrophage to ingest apoptotic cells. For
example, the proinflammatory cytokines granulocyte-macrophage
colony-stimulating factor, IL-1
, TNF-
, and IFN-
can
dramatically upregulate the capacity of human monocyte-derived
macrophages to phagocytose apoptotic neutrophils (58, 74).
In the present studies, we found that in vivo application of soluble
-glucan enhanced spontaneous lymphocyte apoptosis (Fig. 5). Thus,
part of the anti-inflammatory effects of soluble
-glucan treatment
in vivo might result from the enhanced apoptosis of a portion of the
activated lymphocyte population (64, 65, 70). In support of
this idea, Jimenez et al. (36) recently reported that
delayed apoptosis contributed to postoperative systemic inflammatory
response syndrome. Clearly, further studies are necessary to determine
if soluble
-glucan interacts with specific lymphocyte subpopulations
and if soluble
-glucan-induced apoptosis plays a physiologically
important role in resolving inflammatory disease. In contrast to
lymphocytes, there was no evidence for increased spontaneous apoptosis
of monocytes. However, stimulation of monocytes isolated from soluble
-glucan-treated mice with LPS or TSST-1 showed a significantly
increased level of apoptosis. These results suggest a possible role of
soluble
-glucan in enhancing the removal of inflammatory cells from
the sites of inflammation. Clearly, the balance between phagocyte apoptosis and necrosis in inflamed tissues seems to play an important role in the resolution and/or control of inflammation (32, 36, 65).
In summary, our results demonstrate that cytokine release induced by
toxin stimulation of target cells can be manipulated by in vivo
administration of soluble
-glucan in a mouse model. A comparison of
the cytokine profiles of lymphocytes and monocytes isolated from
soluble
-glucan-treated mice, compared to cells from untreated mice,
is summarized in Table 1. In general, the results show a suppressed production of proinflammatory cytokines. Previous studies have demonstrated that part of the anti-inflammatory activity of glucocorticosteroids results from modulation of
proinflammatory cytokine levels (8, 20); however,
glucocorticosteroids also have serious side effects on the body. Thus,
it is possible that the use of immunomodulators, such as soluble
-glucan, to manipulate the production of proinflammatory cytokines
by activated lymphocytes and monocytes will represent a safer
alternative for treating sepsis. These results contribute to the
possible practical application of glucan-derived substances in reducing
the severity of septic shock, especially in situations where
application of anti-cytokine treatment may exacerbate systemic
infection or worsen the outcome in a patient with sepsis
(52).
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Summary of endotoxin- and enterotoxin-stimulated cytokine
responses of leukocytes isolated from soluble -glucan-treated mice
and untreated mice
|
|
 |
ACKNOWLEDGMENTS |
This work was supported in part by USDA/NRICGP grant 9502274, an
Arthritis Foundation biomedical science grant, NIH grant S10 RR11877,
NSF equipment grant DBI-9604797, a grant from the M. J. Murdock
Charitable Trust, USDA Animal Health Formula Funds, the Montana State
University Agricultural Experimental Station, and a grant from the
Slovak Science Grant Agency VEGA (2-5012-98). M.T.Q. is an Established
Investigator of the American Heart Association.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Veterinary Molecular Biology, Montana State University, Bozeman, MT
59717. Phone: (406) 994-5721. Fax: (406) 994-4303. E-mail:
mquinn{at}montana.edu.
Editor:
J. R. McGhee
 |
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Infection and Immunity, January 1999, p. 244-252, Vol. 67, No. 1
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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