From the Engineering Control and Technology Branch
and the ¶ Pathology and Physiology Research Branch, Health Effects
Laboratory Division, National Institute for Occupational Safety and
Health, National Institutes of Health, Morgantown, West
Virginia 26505
Received for publication, February 5, 2001, and in revised form, March 16, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The molecular details of 1 The role of fungi or yeast in organic dust toxic syndrome
has attracted much attention recently. Zymosan A is a cell wall component of yeast, Saccharomyces cerevisiae. Zymosan A
contains 50~57% 1 TNF- Cells and Reagents--
The mouse macrophage cell line,
RAW264.7, was purchased from the American Type Culture Collection
(Manassas, VA). The cells were cultured in RPMI 1640 supplemented with 5% fetal bovine serum (FBS), 2 mM
glutamine, 25 mM HEPES buffer (pH = 7.4), 100 units/ml penicillin and 100 µg/ml streptomycin. The
cells were maintained in 75-cm2 tissue culture flasks at
37 °C in a humidified atmosphere of 5% CO2/95% air.
Specific antibodies against the NF-
The TNF- Zymosan A Stimulation--
For zymosan A stimulation, RAW264.7
cells (0.5 × 106 cells/well) were starved in 1 ml of
0.5% FBS RPMI 1640 medium without phenol red in a 24-well plate for
24 h at 37 °C in a humidified atmosphere of 5%
CO2. Before the experiment, the medium was changed to fresh
0.5% FBS RPMI 1640 medium. The stimulation time and zymosan A
concentration are indicated in the text.
TNF- Nuclear Extraction--
RAW264.7 cells were plated in a 100-mm
culture plate at a density of 8 × 106 cells/plate for
24 h. Then, the cells were starved in RPMI 1640, with 0.5% FBS,
for an additional 24 h. Before the experiment, the medium was
changed to fresh 0.5% FBS RPMI 1640 medium. The cells were treated
with zymosan A or LPS for 4 h. At the end of treatment, the cells
were harvested and treated with 500 µl of lysis buffer (50 mM KCl, 0.5% Nonidet P-40, 25 mM HEPES (pH
7.8), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, 20 µg/ml aprotinin, 100 µM
dithiothreitol) on ice for 4 min. After 1-min centrifugation at
14,000 rpm, the supernatant was saved as a cytoplasmic extract. The
nuclear pellet was washed once with the same volume of buffer without
Nonidet P-40. The nuclear pellet was then treated with 300 µl of
extraction buffer (500 mM KCl, 10% glycerol with the same
concentrations of HEPES, phenylmethylsulfonyl fluoride, leupeptin, aprotinin, and dithiothreitol as the lysis buffer) and pipetted several times. After centrifugation at 14,000 rpm for 5 min, the supernatant was harvested as the nuclear protein extract and
stored at Electrophoretic Mobility Shift Assay (EMSA)--
EMSA assay was
carried out as reported previously (27). Briefly, the DNA-protein
binding reaction was conducted in a 24 µl of reaction mixture
including 1 µg of Poly(dI·dC), 3 µg of nuclear protein extract, 3 µg of bovine serum albumin, 4 × 104 cpm of
32P-labeled oligonucleotide probe, and 12 µl of reaction
buffer (24% glycerol, 24 mM HEPES (pH 7.9), 8 mM Tris-HCl (pH 7.9), 2 mM EDTA, 2 mM dithiothreitol). In some cases, the indicated
amount of double-stranded oligomer was added as a cold competitor. The reaction mixture was incubated on ice for 10 or 20 min (with an antibody) in the absence of a radiolabeled probe. The double-stranded interleukin (IL)-6 NF- Transfection and Luciferase Assay--
A set of TNF-
Statistical Analysis--
Results are given as mean ± S.E.
A paired one-tailed t test (two sample assuming equal
variances) was performed, and the differences were considered
statistically significant at p < 0.05. For multiple comparison, the one way analysis of variance for comparing several treatment groups with one control was used. Statistical analysis was
performed using SigmaStat version 2.0. software (Jandel
Corp.).
Production of TNF- Activation of the TNF- Activation of Transcription Factor NF- Time Course of NF- Inhibition of TNF- Mutation Analysis for NF- Zymosan A is a particulate 1 Conflicting results as to whether or not zymosan activates macrophages
NF- The NF- The activation of NF- In the present study, we have demonstrated that activation of NF- This study provides substantial data for the role of NF- In summary, transcriptional regulation is a major mechanism controlling
cytokine expression. It has been suggested that gene transcription was
activated quickly following 13-
-glucans, a
fungal cell wall component, induced inflammatory responses are not well
understood. In the present study, we conducted a systematic analysis of
the molecular events leading to tumor necrosis factor (TNF)-
production after glucan stimulation of macrophages. We demonstrated
that activation of nuclear factor
B (NF-
B) is essential in
zymosan A (a source of 1
3-
-glucans)-induced TNF-
production in
macrophages (RAW264.7 cells). Zymosan A-induced TNF-
protein
production was associated with an increase in the TNF-
gene promoter
activity. Activation of the TNF-
gene promoter was dependent on
activation of NF-
B. Time course studies indicated that DNA binding
activity of NF-
B preceded TNF-
promoter activity. Inhibition of
NF-
B activation led to a dramatic reduction in both TNF-
promoter activity and TNF-
protein production in the response to zymosan A. Mutation of a major NF-
B binding site (
3) in the gene promoter resulted in a significant decrease in the induction of the gene promoter by zymosan A, while mutation of Egr or CRE sites failed to
inhibit the response to zymosan. Together, these results strongly suggest that NF-
B is involved in signal transduction of
1
3-
-glucans-induced TNF-
expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3-
-glucans (1) and was used as a crude
preparation for 1
3-
-glucans in this study. Inhalation of zymosan
A has been shown to induce an inflammatory response in animal
experiments (2). 1
3-
-Glucans are polymers of
D-glucose, which comprise a major structural component of
fungal cell walls (3). 1
3-
-Glucans have been identified as a
major reticuloendothelial-stimulating component in zymosan (4). A broad
range of cell types can be activated by zymosan A, such as macrophages
(5-7), polymorphonuclear leukocytes (8, 9), and natural killer cells
(10). The interaction of zymosan A with macrophages is generally
considered as the first step in the initiation of an immune response.
Glucan receptors play an important role in mediating binding of zymosan to macrophages (5). The zymosan-induced inflammatory products include
cytokines (11, 12), hydrogen peroxide (13), and arachidonic acid (14).
Pulmonary exposure to zymosan A leads to the infiltration of
polymorphonuclear leukocytes and results in pulmonary inflammation
(2).
1 is a
pro-inflammatory cytokine released from macrophages or activated T
cells in response to microbes or other agents. TNF-
plays a key role
in the initiation of inflammation in the lung and other tissues (15).
TNF-
acts as a chemotactic agent leading to accumulation of
macrophages and polymorphonuclear leukocytes at the inflammatory site
(16). It can prime neutrophils by shortening the lag period of the
respiratory burst (17). Although expression of TNF-
is controlled at
multiple levels, gene transcription is the first and most important
step in the control of TNF-
expression. NF-
B is a critical
transcription factor in the regulation of TNF-
transcription
(18-20). This transcription factor is a heterodimer protein composed
of p65 and p50 in most cases. In addition to TNF-
, NF-
B is also
involved in the regulation of gene transcription for many other
cytokines (21). Nonactivated NF-
B is located in the cytoplasm and is
associated with an inhibitory protein, I-
B (22). I-
B is
phosphorylated and degraded in response to inflammatory stimuli,
leading to the activation of NF-
B. The activated NF-
B
translocates from the cytoplasm into the nucleus, where it binds to
promoter regions of target genes and regulates their transcription.
When target genes are turned on by NF-
B, mRNA synthesis occurs,
and protein expression will follow. Although it has been reported that
1
3-
-glucan is able to activate NF-
B (23) and induce TNF-
production (24, 25), details of the relationship between NF-
B and
TNF-
transcription in response to 1
3-
-glucan remain to be
investigated. This study was designed to explore the details of the
mechanisms regulating TNF-
expression induced by zymosan A at both
the cellular and molecular levels.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B p50 subunit (catalog number
SC-114x, Santa Cruz Biotechnology) and p65 subunit (catalog number
PC137, Oncogene) were used in the supershift assay. The NF-
B
inhibitor, caffeic acid phenethyl ester (CAPE), was purchased from
Biomol Research Laboratory (Plymouth Meeting, PA). Chlorophenolred-
-D-galactopyranoside monosodium salt was
purchased from Roche Molecular Biochemicals. Zymosan A was
obtained from Sigma. Lipopolysaccharide (LPS) was supplied by Difco.
luciferase reporter cells were derived from
reporter-transfected RAW264.7 cells. The cells were transfected with a
TNF-
gene promoter-controlled luciferase plasmid together with a
pcDNA3 plasmid that provides a Geneticin resistance gene. The TNF-
promoter (
1260/+60) controls the luciferase gene expression (26). The transfected cells were first screened by G418 and then by
luciferase activity. The positive cells were cloned, and one of the
clones (clone 6) was used in this study. This clone has been tested for
TNF-
production and luciferase activity in response to LPS. The
results showed that luciferase activity was associated with TNF-
protein production.
ELISA Assay--
After stimulation, the cell cultures
were centrifuged at 1700 rpm for 5 min, and the supernatants were
collected for determination of TNF-
. An ELISA kit from Endogen
(Woburn, MA) was used to determine the TNF-
level in the cell
culture supernatant according to the manufacturer's instructions.
70 °C. The protein concentration was determined using a
BCA protein assay reagent (Pierce).
B probe was labeled with
[32P]ATP (Amersham Pharmacia Biotech) using the T4
kinase (Life Technologies, Inc.). It should be noted that although the
IL-6 NF-
B probe was utilized for the measurement of NF-
B
activation, it is expected that the same results would be obtained
using a TNF-
NF-
B probe. After addition of the radiolabeled
probe, the mixture was incubated for 20 min at room temperature, then
resolved on a 5% acrylamide gel that had been pre-run at 200 V for 30 min with 0.5 × Tri-boric acid EDTA (TBE) buffer. The loaded gel
was run at 200 V for 90 min, then dried and placed on Kodak X-Omat film
(Eastman Kodak Co.) for autoradiography. The film was developed after
an overnight exposure at
70 °C.
reporter
vectors, the TNF-
wild type,
B3-mutated, CRE-mutated, and
Egr-mutated, were gifts from Dr. S. T. Fan at the Scripps Research
Institute (La Jolla, CA) (28). In the wild type vector, a TNF-
gene
promoter fragment (
615/+15) controls the luciferase reporter gene.
The mutant vectors were derived from the wild type vector by point
mutation of the Egr, CRE, or NF-
B (
B3) sites in the promoter,
respectively. The murine macrophage cells (0.5 × 106/well) were plated in a 24-well plate for 16 h,
then were transfected with 0.5 µg of reporter DNA/well using
LipofectAMINE (Life Technologies, Inc.). After transfection, the cells
were washed once in phosphate-buffered saline solution and cultured in
1 ml of the RPMI medium with 0.5% FBS at 37 °C. Zymosan A was added
16 h later, and the cells were harvested at different times for
the reporter assay. The luciferase activity was determined using an
assay kit from Promega (Madison, WI) in combination with a luminometer
(Monolight 3010, Analytical Luminescence Laboratory, Sparks, MD). For
the stable TNF-
reporter cells, 1 × 105 cells/well
were used in a 96-well plate. The cells were lysed in 100 µl of lysis
buffer, and the luciferase activity was determined with a 96-well luminometer.
-Galactosidase was used as an internal control for normalizing
luciferase activity in transient transfection (29).
-Galactosidase activity was determined by colorimetric reaction that was formed by interaction of 100 µl of reaction buffer (80 mM
Na2HPO4, 0.5 M MgCl2,
and 104 mM 2-mercaptoethanol), 20 µl of
chlorophenolred-
-D-galactopyranoside monosodium salt
(80 mM), and 80 µl of cell lysate. The absorbance was
measured at 574 nM after 20 min of incubation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
by Zymosan A-stimulated
Macrophages--
TNF-
is one of the immediate response gene
products from macrophages in the inflammatory response. Therefore,
TNF-
was used as an indicator of macrophage response to zymosan A. The RAW264.7 cells were treated with zymosan A at different
concentrations and for different times. At the end of treatment, the
cell-free supernatant was collected by centrifugation and used for
determination of TNF-
production in an ELISA assay. The results show
that zymosan A induced a substantial TNF-
production in RAW264.7
cells. This induction was dependent on the concentration of zymosan A
(Fig. 1A). A significant
increase in TNF-
production was observed after 24 h of exposure
to 20 µg/ml zymosan A, while a maximum effect (18-fold increase) was
noted at 100 µg/ml zymosan A. The zymosan A-induced TNF-
production was also dependent on stimulation time (Fig. 1B).
The production of TNF-
was significantly increased after a 7-h
exposure to 100 µg/ml zymosan A and reached a maximum by 12.5 h
of exposure. This maximal level was maintained through 40 h of
zymosan A exposure.
View larger version (12K):
[in a new window]
Fig. 1.
Zymosan A-stimulated TNF-
production. The RAW264.7 cells (0.5 × 106
cells/well) were starved for 1 day before addition of zymosan A. The
TNF-
level was measured in the supernatant using a TNF-
ELISA
kit. Values are means ± S.E. of three experiments, "*"
indicates a significant increase from control "-". A, dose-response
curve for zymosan A-induced TNF-
production in RAW264.7 cells.
RAW264.7cells were treated with various concentrations of zymosan A for
24 h. B, time course of TNF-
production in RAW264.7
cells in response to 100 µg/ml zymosan A.
Gene Promoter--
Transcriptional
activity of the TNF-
gene was investigated by analysis of the gene
promoter activity. The TNF-
reporter cells were stimulated with
various concentrations of zymosan A for 8 h. As with TNF-
production, TNF-
promoter response exhibited a relationship to
zymosan A concentration (Fig. 2A). A
significant increase of luciferase activity was observed at 10 µg/ml
zymosan A. A 5.7-fold increase was observed at 100 µg/ml zymosan A,
while an maximum effect (8-fold increase) was noted at 300 µg/ml
zymosan A. The zymosan A-induced TNF-
promoter activation was also
dependent on stimulation time (Fig. 2B). The TNF-
promoter activity was significantly increased at 4 h and reached a
peak at 8 h after exposure to 100 µg/ml zymosan A.
View larger version (24K):
[in a new window]
Fig. 2.
Zymosan A-stimulated TNF-
promoter activity in stable transfected RAW264.7 cells. The
stable transfected RAW264.7 cells (1 × 105
cells/well) were seeded in a 96-well plate for 1 day before addition of
zymosan A. The luciferase activity was measured in the cell lysate
solution. A, dose-response curve for zymosan A-induced
TNF-
promoter activity in stable transfected RAW264.7 cells.
RAW264.7 cells were treated with various concentrations of zymosan A
for 8 h. B, time course of TNF-
promoter activity in
stable transfected RAW264.7 cells in response to 100 µg/ml of zymosan
A. Values are means ± S.E. of four experiments, "*" indicates
significantly higher than control "-".
B--
NF-
B is a major
activator for TNF-
transcription in macrophages. It is not clear if
NF-
B regulates TNF-
transcription in response to zymosan A. To
investigate the role of NF-
B, the DNA binding activity of NF-
B
was examined in the nuclear extract of zymosan A-treated cells. The
cells were treated with 100 µg/ml zymosan A for 4 h, and
the nuclear protein was extracted as stated under "Materials and
Methods." The results show that the nuclear proteins from the control
cells formed a typical pattern of NF-
B bands (Fig.
3A, lane 1), and
the binding activities were remarkably increased (2.8-fold) by zymosan
A stimulation (Fig. 3A, lane 2). The nature of
the DNA-protein complexes was determined in the supershift assay and
the competition assay (Fig. 3A, lanes 3-7). The
supershift result shows that the upper band was formed by a heterodimer
of p65 and p50 subunits (Fig. 3A, lane 5), and
the lower band was formed by a homodimer of p50 subunits (Fig.
3A, lane 6). The gel shift results in Fig.
3A were quantified using a densitometer (Fig.
3B). The oligonucleotide competition result demonstrates
that NF-
B complexes were formed specifically by interaction between
the NF-
B binding probe and nuclear proteins, since DNA binding was
inhibited by cold NF-
B but not by cold AP-1 or ATF-1 antibodies.
Both the upper band and lower bands were enhanced by zymosan A. The
upper band (p65 subunit) is thought to be critical for induction of
TNF-
-dependent genes (20).
View larger version (36K):
[in a new window]
Fig. 3.
DNA binding activity of transcription factor
NF- B in the nuclear extract of RAW264.7
cells. The DNA binding activity of NF-
B in the nuclear extract
was determined using the EMSA gel shift assay (A) and the
densities of the NF-
B bands measured and normalized for gel loading
(B). The normalization was done by the sum of density of
NF-
B p65/p50 and p50/p50 bands divided by the nonspecific band.
A, characterization of NF-
B complexes in the
oligonucleotide competition and antibody supershift assays. Lane
1 served as a control for the oligonucleotide competition assay.
Lanes 2-7 are nuclear proteins from cells treated with
zymosan A (100 µg/ml) for 4 h. The unlabeled NF-
B probe (0.2 µg) was used in lane 3 as a specific competitor. The same
amount of unlabeled AP-1 probe was used in lane 4 for
nonspecific competition. The antibodies against the p65 or p50 subunit
of NF-
B protein were added in lane 5 or 6 to
confirm the nature of DNA-protein complexes. Lane 7 contained 1 µg of ATF-1 antibody to serve as a nonspecific
antibody.
B Activation--
The above results
demonstrate that zymosan induced both NF-
B DNA binding and
activation of the TNF-
gene promoter. If NF-
B is responsible for
activation of the promoter activity in response to zymosan, its
activation should precede the promoter activity. To examine this
hypothesis, the time course of NF-
B binding activity was
investigated in the nuclear proteins from zymosan-treated cells (Fig.
4). The result shows that NF-
B was
significantly activated at 2 h after zymosan A stimulation. The
NF-
B binding activity continuously increased and reached a peak
around 8 h after zymosan A treatment (Fig. 4A). The
binding intensity was quantified by densitometry, and the results are
shown in Fig. 4B. As this time course precedes the time
course of the gene promoter activation (Fig. 2B), the
results indicate that NF-
B might be responsible for activation of
the TNF-
gene promoter.
View larger version (37K):
[in a new window]
Fig. 4.
Time course of DNA binding activity of
transcription factor NF- B in the nuclear
extract of RAW264.7 cells. Macrophages were treated with 100 µg/ml zymosan A for various times, and the DNA binding activity of
NF-
B in the nuclear extract was determined using the EMSA gel shift
assay (A), and the densities of the NF-
B bands were
normalized (B). The normalization was done by the sum of the
density of NF-
B p65/p50 and p50/p50 bands divided by the
nonspecific band.
Promoter by the NF-
B Inhibitor
CAPE--
To explore the role of NF-
B in activation of TNF-
transcription further, the NF-
B inhibitor CAPE was used in the
study. This NF-
B inhibitor has been reported to prevent the
translocation of the p65 subunit of NF-
B from the cytoplasm to the
nucleus (30), resulting in a decrease in DNA binding activity of
NF-
B. The inhibition is specific for NF-
B and does not affect the
DNA binding activities of other transcription factors, including AP-1, Oct-1, and TFIID (30). The cells were pre-treated with CAPE for 1 h, then followed by 4-h zymosan A exposure. DNA binding activity of
NF-
B was examined in EMSA. The results show that CAPE significantly
suppressed the zymosan A-induced activation of NF-
B (Fig.
5, A and B). The
upper band (p65/p50 heterodimer), which is critical for induction of
TNF-
-dependent genes, was completely inhibited by CAPE
(Fig. 5A, lane 3). The RAW264.7 cells show a
normal NF-
B induction when using a positive control LPS (Fig.
5A, lane 4). TNF-
promoter response was also
assayed after the CAPE treatment. The TNF-
luciferase cells were
used to test inhibitory effect of CAPE. The results demonstrate that
CAPE significantly reduced (by 80%) zymosan A-induced promoter
activity (Fig. 5C). In line with this inhibition, the
zymosan-induced TNF-
expression was also completely abolished by
CAPE (Fig. 5D). These results further support a role of
NF-
B in zymosan-induced TNF-
production.
View larger version (41K):
[in a new window]
Fig. 5.
NF- B inhibitor,
CAPE, decreased DNA binding activity of NF-
B,
TNF-
promoter activation, and
TNF-
expression. A and
B, CAPE inhibits DNA binding activity of NF-
B.
A: lane 1, untreated RAW264.7 cells; lane
2, cells treated for 4 h with 100 µg/ml zymosan
A, which show enhanced NF-
B binding activity to the DNA;
lane 3, cells pre-treated with CAPE (10 µg/ml) for 1 h, then exposed to zymosan for 4 h; lane 4, the
positive control LPS (10 µg/ml). C, CAPE inhibits TNF-
promoter activity. The TNF-
reporter cells were pre-treated with
CAPE (10 µg/ml) for 1 h before addition of 100 µg/ml zymosan A
into the culture medium. The luciferase activity was measured in the
cell lysate solution at 8 h after adding zymosan A. Values are
means ± S.E. of four experiments. "*" indicates a significant
increase from the control. "-" indicates a significant decrease
from the zymosan A-induced level. D, CAPE inhibits TNF-
production. RAW264.7 cells were pre-treated with CAPE (10 µg/ml) for
1 h before addition of 100 µg/ml zymosan A into the culture
medium. The TNF-
production was measured 24 h after zymosan A
stimulation. Values are means ± S.E. of three experiments.
"*" indicates a significant increase from control. "-"
indicates a significant decrease from the zymosan A-induced
level.
B Binding Site--
If NF-
B is
required for TNF-
transcription, mutation of the NF-
B binding
site in the gene promoter should lead to loss of the promoter response
to zymosan A. To test this hypothesis, mutated TNF-
promoters were
employed in the transient transfection assay. The result shows that
zymosan A generated a 4-fold induction with the wild type TNF-
promoter (Fig. 6A), while only
a 1.5-fold induction was observed with the
B3-mutated TNF-
promoter in which the major NF-
B binding site (
3) was mutated to
inhibit the NF-
B binding activity (Fig. 6B). In contrast,
when the CRE or Egr site was mutated in the TNF-
promoter, no
reduction in zymosan A-induced TNF-
promoter activity was observed
(Fig. 6, C and D). These results demonstrate that
the NF-
B binding site (
B3) is critical for activation of the
TNF-
promoter by zymosan A.
View larger version (27K):
[in a new window]
Fig. 6.
Zymosan A-induced promoter activity of
TNF- in the transient transfection assay and
the dependence of TNF-
gene expression on
B, Egr, and CRE sites in the promoter. The
wild type or
B-, Egr-, or CRE-mutated TNF-
luciferase reporters
were transfected into the cells. The cells were treated with zymosan A
(100 µg/ml) for 4 h. The reporter activity in the cell lysate
was determined using a luminometer, and the reading was normalized by
-galactosidase activity. Values are means ± S.E. of three
experiments. "+" indicates a significant increase from
the control. "-" indicates a value significantly less than the wild
type.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3-
-glucan that induces immune
responses by activating macrophages. Macrophages play an essential role
in orchestrating the inflammatory response by the selective production
of cytokines. In vitro studies have demonstrated that 1
3-
-glucans induce secretion of both IL-1 and TNF-
in mouse peritoneal macrophages (31) and in human monocytes (32, 33). Northern
blot analysis showed that TNF-
mRNA was increased within 30 min,
peaked at 2 h, and remained elevated for at least 8 h after
exposure of human monocytes to 1
3-
-glucans (32). Zymosan A
activity was mediated by
-glucan receptors (32). In vivo study shows that 1
3-
-glucans are able to induce a transient increase of both IL-1 and IL-6 in mouse blood (34). In addition to
IL-1, TNF-
, and IL-6, 1
3-
-glucans also induce expression of
IL-8 (11). These cytokines may share a common mechanism of induction in
the response to 1
3-
-glucans. It has been shown in the literature
that many human cytokines are regulated by NF-
B (21), which includes
IL-1, IL-6, IL-8, and TNF-
. NF-
B binding sites have been
identified in the promoter regions of these cytokine genes. Therefore,
we hypothesize that NF-
B might be one of the major mediators of
zymosan A signals for induction of inflammatory responses.
B signal transduction pathway have been reported. Tran-Thi
et al. (35) reported that zymosan is incapable of activating NF-
B in rat liver macrophages. However, 1
3-
-glucans have been shown to activate NF-
B in the human promonocytic cell line U937 (23). This agrees with results reported in the present study using
RAW264.7 mouse peritoneal macrophages. In addition, our laboratory has
observed NF-
B activation in response to 1
3-
-glucans with
NR8383 cells, an alveolar macrophage cell line from the
rat.2 Furthermore, the
Tran-Thi et al. (35) study was focused on LPS-induced
NF-
B and AP-1 activation rather than zymosan-induced responses.
Therefore, the dose and exposure time may not have been optimized for
zymosan. Last, 1
3-
-glucans activation of rat liver macrophage has
been reported in the literature. Indeed, Adachi et al. (36)
described that 1
3-
-glucans could enhance the production of
cytokines and nitric oxide in these macrophages. Although they did not
study the activation of NF-
B, it is quite likely that NF-
B was
activated during 1
3-
-glucans exposure, since NF-
B is an
upstream signal for these inflammation responses.
B signaling system has been considered an evolutionarily
conserved system that can operate in divergent genes in many different
species (37). NF-
B is inducible. When cells receive an inflammatory
stimulation, NF-
B will be activated and up-regulate the
transcription of cytokine production. Transcriptional regulation is a
major mechanism controlling cytokine expression. Initiation of
transcription is determined by the promoter region in a cytokine gene.
When the
B motif in the promoter DNA is occupied by NF-
B, there
is initiation of transcription. The consensus DNA sequence for the
NF-
B motif is, 5'-GGGRNNYYCC-3'. The DNA sequence of NF-
B binding
site in IL-6 is 5'-GGGATTTTCC-3' and in TNF-
is 5'-GGGGCTTTCC-3',
5'-GGGAAAGCCC-3', and 5'-GGGAATTCAC-3' (38). The present study examines
the relationship between zymosan-stimulated NF-
B activation and
TNF-
production. The results support a model for transcriptional
regulation of cytokines induced by 1
3-
-glucans.
B is commonly associated with the degradation
of I-
B. The I-
B family are NF-
B inhibitory units that contain
ankyrin repeat domains that bind to NF-
B and mask the nuclear
localization signal (21). Following 1
3-
-glucans stimulation, I-
B is phosphorylated and degraded, unmasking nuclear localization signals and allowing NF-
B to be transported to the cell nucleus. NF-
B then binds to promoter regions of target genes and regulates their transcription. Whether or not zymosan activation of NF-
B is
done through the degradation of I-
B is unclear. A decrease of
I-
B level was associated with
Betafectin®-induced stimulation of mouse BMC2.3
macrophage cells (39). However, Bondeson et al. (40)
reported that overexpression of I-
B
by adenoviral gene
transfection had no effect on zymosan-induced IL-8, IL-1
, or TNF-
levels. This may imply that zymosan activates NF-
B through a pathway
other than I-
B
degradation (41). However, the lack of direct
evidence on I-
B activity after zymosan exposure suggests that
further study is needed to verify the above hypothesis.
B
is essential in zymosan A (a source of 1
3-
-glucans)-induced TNF-
production by RAW264.7 macrophages. Zymosan A increased TNF-
production in a time- and concentration-dependent manner (Fig. 1, A and B). The optimum dose for induction
was about 100 µg/ml. This dose was approximately the optimal dose for
TNF-
promoter activation (Fig. 2A). We then established
the time course for TNF-
production and TNF-
promoter activation
(Figs. 1B and 2B). The results show that TNF-
promoter activation was significantly increased 4 h after zymosan
A exposure and peaked at 8 h. TNF-
promoter activation preceded
TNF-
production (significant increase at 7 h with a peak at
12.5 h). TNF-
promoter activation was, in turn, preceded by
NF-
B/DNA binding, which was significantly elevated 2 h after
zymosan A treatment and peaked at 8 h. Pre-treatment of macrophage
cells with a NF-
B inhibitor led to a decrease in DNA binding
activity of the NF-
B p65/p50 heterodimer (Fig. 5, A and
B), which led to a subsequent decrease in TNF-
promoter activity (Fig. 5C) and a suppression of TNF-
production
(Fig. 5D). These results suggest that zymosan A is able to
induce TNF-
expression by NF-
B-dependent activation
of gene transcription.
B in the
transcriptional activation of TNF-
by zymosan A. 1) Zymosan A
induced the activation of NF-
B. The DNA binding activity of NF-
B
in the nuclear extract was enhanced (2.8-fold) after zymosan A
treatment. 2) The time course of DNA binding activity of NF-
B preceded the promoter activation of TNF-
. This suggests that the
dynamic change of the TNF-
promoter activity results from a change
in the DNA binding activity of NF-
B. 3) Inhibition of NF-
B
activation decreased zymosan A-stimulated TNF-
promoter activity by
80%. 4) Removal of the NF-
B binding site led to inhibition of the
TNF-
promoter activation. The importance of NF-
B in TNF-
transcription was investigated with mutation of the major NF-
B binding site (
B3). Mutation studies show that the promoter response to zymosan A was dramatically reduced (decreased by 80%) after mutation, while mutation of the Egr or CRE site had no effect on
promoter activation (Fig. 6). Taken together, these data strongly support the hypothesis that NF-
B mediates zymosan A-induced TNF-
transcription and TNF-
production in macrophages.
3-
-glucans exposure (32). Initiation
of transcription is determined by the promoter region in a cytokine
gene. NF-
B is an activator protein for many cytokine gene promoters,
including IL-1, IL-6, IL-8, and TNF-
(22). We hypothesize that
NF-
B is a major mediator of zymosan A signals for induction of these
cytokines. The present study used the TNF-
gene as a model for
analysis of the transcriptional regulation of 1
3-
-glucans
response cytokines. The results demonstrate that the activation of
NF-
B is essential for zymosan A-induced TNF-
production in macrophages.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Held a National Research Council-NIOSH Research Associateship while this work was performed. To whom correspondence should be addressed: ECTB, HELD, NIOSH, 1095 Willowdale Rd., Morgantown, WV 26505. Tel.: 304-285-6225 Fax: 304-285-6265; E-mail: syoung@cdc.gov.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M101111200
2 S.-H. Young, J. Ye, D. G. Frazer, X. Shi, and V. Castranova, unpublished result.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor
necrosis factor;
NF-B, nuclear factor
B;
FBS, fetal bovine serum;
CAPE, caffeic acid phenethyl ester;
LPS, lipopolysaccharide;
ELISA, enzyme-linked immunosorbent assay;
EMSA, electrophoretic mobility shift
assay;
IL, interleukin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | DiCarlo, F. J., and Fiore, J. V. (1957) Science 127, 756-757 |
2. | Robinson, V. A., Frazer, D. G., Afshari, A. A., Goldsmith, W. T., Olenchock, S., Whitmer, M. P., and Castranova, V. (1996) in Proceedings of the 20th Cotton and Organic Dust Research Conferences (Jacobs, R. R. , Wakelyn, P. J. , and Rylander, R., eds) , pp. 356-360, National Cotton Council, Nashville, TN |
3. | Manners, D. J., Masson, A. J., and Patterson, J. C. (1973) Biochem. J. 135, 19-30[Medline] [Order article via Infotrieve] |
4. |
Riggi, S. J.,
and Di Luzio, N. R.
(1961)
Am. J. Physiol.
200,
297-300 |
5. | Tapper, H., and Sundler, R. (1995) Biochem. J. 306, 829-835[Medline] [Order article via Infotrieve] |
6. | Tennent, R. J., and Donald, K. J. (1976) J. Reticuloendothel. Soc. 19, 269-280[Medline] [Order article via Infotrieve] |
7. | Sorenson, W. G., Shahan, T. A., and Simpson, J. (1998) Ann. Agric. Environ. Med. 5, 1-7[Medline] [Order article via Infotrieve] |
8. | Adachi, Y., Okazaki, M., Ohno, N., and Yadomae, T. (1997) Mediat. Inflamm. 6, 251-256[CrossRef] |
9. | Morikawa, K., Takeda, R., Yamazaki, M., and Mizuno, D. (1985) Cancer Res. 45, 1496-1501[Abstract] |
10. | Duan, X., Ackerly, M., Vivier, E., and Anderson, P. (1994) Cell. Immunol. 157, 393-402[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Noble, P. W.,
Henson, P. M.,
Lucas, C.,
Mora-Worms, M.,
Carre, P. C.,
and Riches, D. W.
(1993)
J. Immunol.
151,
979-989 |
12. | Sakurai, T., Kaise, T., Yadomae, T., and Matsubara, C. (1997) Eur. J. Pharmacol. 334, 255-263[CrossRef][Medline] [Order article via Infotrieve] |
13. | Chiba, N., Ohno, N., Terui, T., Adachi, Y., and Yadomae, T. (1996) Pharm. Pharmacol. Lett. 6, 12-15 |
14. | Daum, T., and Rohrbach, M. S. (1992) FEBS Lett. 309, 119-122[CrossRef][Medline] [Order article via Infotrieve] |
15. | Driscoll, K. E., Carter, J. M., Hassenbein, D. G., and Howard, B. (1997) Environ. Health Perspect. 105 Suppl. 5, 1159-1164[Medline] [Order article via Infotrieve] |
16. |
Ming, W. J.,
Bersani, L.,
and Mantovani, A.
(1987)
J. Immunol.
138,
1469-1474 |
17. | Humbert, J. R., and Winsor, E. L. (1990) Am. J. Med. Sci. 300, 209-213[Medline] [Order article via Infotrieve] |
18. |
Bohuslav, J.,
Kravchenko, V. V.,
Parry, G. C.,
Erlich, J. H.,
Gerondakis, S.,
Mackman, N.,
and Ulevitch, R. J.
(1998)
J. Clin. Invest.
102,
1645-1652 |
19. | Carpentier, I., Declercq, W., Malinin, N. L., Wallach, D., Fiers, W., and Beyaert, R. (1998) FEBS Lett. 425, 195-198[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Beg, A. A.,
and Baltimore, D.
(1996)
Science
274,
782-784 |
21. |
Blackwell, T. S.,
and Christman, J. W.
(1997)
Am. J. Respir. Cell Mol. Biol.
17,
3-9 |
22. | Baeuerle, P. A., and Henkel, T. (1994) Annu. Rev. Immunol. 12, 141-179[CrossRef][Medline] [Order article via Infotrieve] |
23. | Battle, J., Ha, T., Li, C., Della Beffa, V., Rice, P., Kalbfleisch, J., Browder, W., and Williams, D. (1998) Biochem. Biophys. Res. Commun. 249, 499-504[CrossRef][Medline] [Order article via Infotrieve] |
24. | Suzuki, T., Ohno, N., Chiba, N., Miura, N., Adachi, Y., and Yadomae, T. (1996) J. Pharm. Pharmacol. 48, 1243-1248[Medline] [Order article via Infotrieve] |
25. | Ohno, N., Miura, N. N., Chiba, N., Adachi, Y., and Yadomae, T. (1995) Biol. Pharm. Bull. 18, 1242-1247[Medline] [Order article via Infotrieve] |
26. |
Baer, M.,
Dillner, A.,
Schwartz, R. C.,
Sedon, C.,
Nedospasov, S.,
and Johnson, P. F.
(1998)
Mol. Cell. Biol.
18,
5678-5689 |
27. |
Ye, J.,
Ghosh, P.,
Cippitelli, M.,
Subleski, J.,
Hardy, K. J.,
Ortaldo, J. R.,
and Young, H. A.
(1994)
J. Biol. Chem.
269,
25728-25734 |
28. |
Yao, J.,
Mackman, N.,
Edgington, T. S.,
and Fan, S. T.
(1997)
J. Biol. Chem.
272,
17795-17801 |
29. | Alam, J., and Cook, J. L. (1990) Anal. Biochem. 188, 245-254[Medline] [Order article via Infotrieve] |
30. |
Natarajan, K.,
Singh, S.,
Burke, T. R., Jr.,
Grunberger, D.,
and Aggarwal, B. B.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9090-9095 |
31. | Ohno, N., Hashimoto, T., Adachi, Y., and Yadomae, T. (1996) Immunol. Lett. 52, 1-7[CrossRef][Medline] [Order article via Infotrieve] |
32. | Abel, G., and Czop, J. K. (1992) Int. J. Immunopharmcol. 14, 1363-1373[CrossRef][Medline] [Order article via Infotrieve] |
33. | Doita, M., Rasmussen, L. T., Seljelid, R., and Lipsky, P. E. (1991) J. Leukoc. Biol. 49, 342-351[Abstract] |
34. | Kondo, Y., Kato, A., Hojo, H., Nozoe, S., Takeuchi, M., and Ochi, K. (1992) J. Pharmacobio-Dyn. 15, 617-621[Medline] [Order article via Infotrieve] |
35. | Tran-Thi, T. A., Decker, K., and Baeuerle, P. A. (1995) Hepatology 22, 613-619[Medline] [Order article via Infotrieve] |
36. | Adachi, Y., Ohno, N., and Yadomae, T. (1998) Biol. Pharm. Bull. 21, 278-283[Medline] [Order article via Infotrieve] |
37. | Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16, 225-260[CrossRef][Medline] [Order article via Infotrieve] |
38. | Baeuerle, P. A. (1991) Biochim. Biophys. Acta 1072, 63-80[CrossRef][Medline] [Order article via Infotrieve] |
39. | Adams, D., Nathans, R., Pero, S., Sen, A., and Wakshull, E. (2000) J. Cell. Biochem. 77, 221-233[Medline] [Order article via Infotrieve] |
40. |
Bondeson, J.,
Browne, K. A.,
Brennan, F. M.,
Foxwell, B. M.,
and Feldmann, M.
(1999)
J. Immunol.
162,
2939-2945 |
41. | Imbert, V., Rupec, R., Livolsi, A., Pahl, H., Traenckner, E., Mueller- Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P., and Peyron, J. (1996) Cell 86, 787-798[Medline] [Order article via Infotrieve] |