1 Combined Program in Pulmonary
and Critical Care Medicine, Regulation of eotaxin expression was
investigated in U-937 cells, a human monocyte-like cell line. Eotaxin
mRNA was induced by tumor necrosis factor-
dexamethasone; protein kinase C; nuclear factor- EOSINOPHIL RECRUITMENT into tissues is known to be a
feature of parasitic infection and allergic inflammation (3, 32). Advances in eosinophil biology have included the discovery of cytokines
that are chemotactic for eosinophils: regulated on activation normal T
cell expressed and secreted (RANTES), macrophage
inflammatory protein-1 Monocytic cells are known to be an important source of chemokines such
as IL-8 and MCP-1 (5, 21). Alveolar macrophages are the dominant
mononuclear cell form in the air spaces and are an important source of
cytokines that are associated with airway inflammation and allergic
disease of the airways (4). Recent reports have demonstrated that, in
the inflamed human airway, eotaxin is produced by both epithelial cells
and mononuclear cells present in the subepithelial layer (16, 24). We
have recently reported the regulatory mechanisms of eotaxin expression
in human lung epithelial cells (17), but the regulation of eotaxin
expression in monocytic cells has not yet been elucidated. We therefore
studied the mechanisms by which eotaxin expression is regulated in a
human monocyte-like cell line.
The mechanisms that govern the regulation of eotaxin expression in
inflammatory cells are relevant to the pathophysiology of eosinophilic
inflammation. Differences in the mechanisms that drive eotaxin
expression at distinct tissue loci may be important for the creation of
chemotactic gradients of eotaxin that govern eosinophil distribution in
the tissues. We have demonstrated that proinflammatory cytokines
including IL-1 U-937 cell culture. U-937 cells, which
are derived from human histiocytic lymphoma cells and have
monocyte-like characteristics (1), were obtained from the American Type
Culture Collection (Manassas, VA). The cells were cultured in RPMI 1640 with 10% heat-inactivated fetal bovine serum (FBS) at a concentration
of 2 × 106 cells/ml. The
cells were then stimulated with geometrically increasing doses of
TNF- Human monocyte and epithelial cell
culture. Peripheral blood mononuclear cells (PBMCs)
were isolated from the heparinized venous blood of three healthy
platelet donors by density gradient centrifugation with Histopaque 1077 (Sigma, St. Louis, MO). This procedure yielded ~5 × 108 cells from each donor, with a
PBMC purity of >98%. None of the subjects had allergic disease or
peripheral blood eosinophilia (eosinophil percentages were <5%), and
all gave informed written consent with the prior approval of the
appropriate institutional review board. PBMCs were cultured in RPMI
1640 with 10% type AB human serum (Sigma) on 10-cm culture plates
(Falcon 3003, Becton Dickinson Laboratories, Lincoln Park, NJ)
overnight at a concentration of 5 × 106 cells/ml. After nonadherent
cells were removed, adherent cells were washed three times with PBS and
designated as monocytes. Monocyte purity was judged to be >80% by
microscopic examination.
A549 cells, derived from a lung adenocarcinoma with the alveolar type
II cell phenotype, were obtained from the American Type Culture
Collection. The cells were cultured in F-12K medium with 10% FBS.
Twenty-four hours before stimulation when the cells were grown to
confluence, the medium was exchanged for an identical formulation not
containing FBS.
Monocytes and A549 cells were cultured in the absence or presence of
TNF- TNF- Neutralization of
TNF- To confirm the neutralizing ability of the antibody for TNF- RNA analysis. Total RNA was isolated
from freshly harvested cells by guanidinium-thiocyanate-phenol
chloroform extraction (Stratagene, La Jolla, CA). For Northern
analysis, 20 µg of total RNA were subjected to gel electrophoresis on
a formaldehyde-2% agarose gel and transferred to a nylon membrane
(Schleicher & Schuell, Keene, NH). After ultraviolet cross-linking, the
membrane was hybridized at 68°C in ExpressHyb Hybridization
Solution (Clontech, Palo Alto, CA) with a
32P-labeled 0.35-kb cDNA probe
containing the entire coding region of the human eotaxin gene (10), a
0.8-kb cDNA probe of the 10-kDa IFN- Nuclear extract preparation and electrophoretic
mobility shift assay. Nuclear extracts were prepared as
previously described by Takeshita et al. (29). U-937 cells were
cultured for 2 or 8 h in the absence or presence of TNF- Electrophoretic mobility shift assay (EMSA) was performed by standard
methods (29). Briefly, 2 µg of nuclear extract under each condition
were incubated for 20 min at room temperature in 20 µl of binding
buffer [10 mM Tris · HCl (pH 7.5), 1 mM EDTA, 1 mM Immunocytochemical staining. Cytospin
slides were prepared with U-937 cells cultured under the following
conditions: unstimulated (8 h), TNF- Time courses of eotaxin mRNA expression by
TNF-
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(TNF-
; 0.1-100
ng/ml) and phorbol 12-myristate 13-acetate (PMA; 0.01-1 µM).
PMA-induced eotaxin mRNA expression was of greater magnitude and was
maximal at a later time point than TNF-
-induced expression (16 h vs.
2 h after stimulation), which was consistent with eotaxin protein
expression detected by immunocytochemistry. Dexamethasone (0.01-10
µM) decreased eotaxin mRNA expression in both TNF-
- and
PMA-stimulated U-937 cells. PMA-induced eotaxin mRNA expression was
inhibited by cycloheximide (10 µg/ml), whereas TNF-
-induced
expression was not. The protein kinase C (PKC) inhibitor staurosporine
(10-50 nM) inhibited PMA-induced eotaxin mRNA expression, whereas
TNF-
-induced expression was enhanced by this reagent. These results
suggest that eotaxin expression can be induced by more than one
mechanism: the PMA-triggered pathway is mediated by PKC activation and
requires new protein synthesis, whereas the TNF-
-triggered pathway
is independent of PKC and protein synthesis. TNF-
- and PMA-induced
pathways are both associated with nuclear factor-
B, because its
binding activity was enhanced in the presence of these stimuli, and
both pathways were limited by its inhibitor, diethyldithiocarbamate.
B; cycloheximide
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(MIP-1
), monocyte chemoattractant protein
(MCP)-3, MCP-4, and interleukin (IL)-16 (9, 13, 26). Most of these cytokines are not specific for eosinophils because they act through receptor systems that are present on a variety of cell types (13, 14).
Eotaxin is a unique member of the C-C family of chemokines that
selectively recruits eosinophils into the skin of guinea pigs and
primates (7, 24). The mechanism of this selective effect on eosinophils
is thought to relate to the high affinity of eotaxin for the chemokine
receptor-3 (CCR-3), which is expressed predominantly on eosinophils (8,
14).
and tumor necrosis factor-
(TNF-
) induce
eotaxin expression in human lung epithelial cells, which is enhanced in
the presence of interferon-
(IFN-
) and suppressed by
dexamethasone (17). In addition to these cytokines, lipopolysaccharide
(LPS) and phorbol 12-myristate 13-acetate (PMA) are known to be potent
inducers of cytokines in monocytic cells (21, 22). PMA is also a
well-established protein kinase C (PKC) activator that can modulate
cytokine expression (2). To determine differences among airway
epithelial cells and other mononuclear cell types known to produce
eotaxin in the human airways, we examined the mechanisms by which these
cytokines and agents influence eotaxin expression in U-937 cells.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, PMA, IL-1
, IFN-
, IL-4, or LPS. In experiments involving
dexamethasone, cycloheximide, staurosporine, or diethyldithiocarbamate (DETC), the agents were added 30 min before cell stimulation. Viability
was >95% in the cells treated with dexamethasone, cycloheximide, staurosporine, or DETC after culture for 16 h. In the experiments with
cycloheximide, >90% of radiolabeled methionine uptake was inhibited.
In the time-course experiments, cells were harvested 1, 2, 4, 8, 16, 24, and 48 h after stimulation. In concentration-response studies, the
cells were harvested at the determined times of peak expression, which
were 2 h after stimulation with TNF-
and 16 h after stimulation with
PMA. All cell stimulation experiments were performed at least in
duplicate.
(10 ng/ml) and PMA (0.1 µM) for 4 h. In experiments involving
cycloheximide (10 µg/ml), staurosporine (50 nM), or DETC (1 mM), the
agents were added 30 min before cell stimulation. Viability was >95%
in the cells treated with these reagents for 4 h. In the experiments
with cycloheximide, >90% of radiolabeled methionine uptake was
inhibited in PBMCs and A549 cells. All cell stimulation experiments
were performed at least in duplicate.
immunoassay. TNF-
concentrations in the culture supernatant of U-937 cells (2 × 106/ml) stimulated with 0.1 µM
PMA were measured by a sandwich enzyme-linked immunosorbent assay (R&D
Systems, Minneapolis, MN). The minimum detectable concentration was 4.4 pg/ml. The culture supernatant was collected 0, 1, 2, 4, 8, 16, 24, and
48 h after stimulation with PMA (n = 4).
. U-937 cells were stimulated with
0.1 µM PMA for 16 h in the absence or presence of an anti-human TNF-
neutralizing antibody or a control monoclonal antibody (both mouse IgG1, R&D Systems) in
duplicate. The antibodies (1 µg/ml) were added to the cells
simultaneously with PMA. The 50% effective neutralizing dose
(ND50) of this
neutralizing antibody is reported in the manufacturer's instructions
to be 0.02-0.04 µg/ml for 0.25 ng TNF-
/ml in murine L929
cells.
-induced
eotaxin expression in U-937 cells, the neutralizing or control antibody
(1 µg/ml) was incubated with TNF-
(1 or 10 ng/ml) for 1 h at
37°C. After incubation, the cells were stimulated with the mixture
for 2 h. Isolated total RNA was then subjected to Northern analysis.
-inducible protein
(IP-10) (18), or a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe (Clontech). The membranes were washed
for 10 min at room temperature in 2× saline-sodium citrate (SSC;
1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0)-0.05% SDS and then for 20 min at 50°C in 0.2× SSC-0.1% SDS. To
control for RNA loading, the hybridization signal obtained for eotaxin or IP-10 was normalized to that for GAPDH in each sample.
(10 ng/ml)
and PMA (0.1 µM). After culture, the cells were washed with PBS and
incubated with buffer A [10 mM
HEPES (pH 7.9), 5 mM dithiothreitol (DTT), 0.3 M sucrose, 0.1 mM EGTA,
and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] containing 1 µg/ml of antipain, aprotinin, chymostatin, leupeptin, and pepstatin A
(Sigma) on ice for 15 min. After centrifugation, the cells were
resuspended in 1 ml of buffer A
containing the protease inhibitors and were subjected to Dounce
homogenization (20 strokes). The homogenates were microcentrifuged for
30 s, and nuclei were resuspended in 200 µl of
buffer B [20 mM HEPES (pH 7.9),
0.5 mM DTT, 5 mM MgCl2, 300 mM
KCl, 25% glycerol, 0.2 mM EGTA, and 0.5 mM PMSF] and rocked at
4°C for 30 min. After microcentrifugation, the supernatants were
dialyzed against 200 ml of buffer D
[20 mM HEPES (pH 7.9), 0.5 mM DTT, 100 mM KCl, 20% glycerol, 0.2 mM EDTA, and 0.5 mM PMSF] at 4°C overnight. After
microcentrifugation, protein concentrations of the supernatants were
measured by the Coomassie blue protein assay (Pierce, Rockford, IL).
-mercaptoethanol, 4% glycerol, and 40 mM NaCl] containing 50 pg of
-32P-labeled NF-
B
consensus oligonucleotide probe [35,000 counts/min (cpm),
5'-AGTTGAGGGGACTTTCCCAGGC-3', Santa Cruz Biotechnology, Santa Cruz, CA] and 0.5 µg of poly(dI-dC) (Pharmacia Biotech, Piscataway, NJ). A competition assay was performed by the addition of
100-fold molar excess unlabeled probe to nuclear extracts 10 min before
the binding reaction with the labeled probe was begun. DNA-protein
complexes were separated in a 4% polyacrylamide gel and analyzed by
autoradiography.
stimulated (10 ng/ml, 8 h),
unstimulated (48 h), and PMA stimulated (0.1 µM, 48 h). For eotaxin
identification, a rabbit polyclonal anti-human eotaxin antibody was
used. The cytospins were fixed in 4% paraformaldehyde for 10 min and
then treated with trypsin for 5 min. Nonspecific immunoglobulin binding was blocked with 10% normal goat serum. The primary rabbit polyclonal antibody, diluted 1:400 in PBS with 2% bovine serum albumin, was applied to the samples and incubated at 4°C overnight. The slides were then incubated in the secondary antibody (biotinylated goat anti-rabbit IgG, Vector Laboratories, Burlingame, CA) at 4°C for 2 h. Endogenous peroxidase activity was quenched with methanol containing
1% hydrogen peroxide. Avidin-biotin complex standard (Vector Laboratories) was applied to the samples and incubated at room
temperature for 1 h. Immunopositivity was localized with the chromagen
diaminobenzidine (0.025%) in PBS and 0.1% hydrogen peroxide. As a
negative control, rabbit IgG (Vector Laboratories) was substituted for
the primary antibody. The rabbit polyclonal anti-human eotaxin antibody
reacted strongly to 100 ng of human eotaxin but did not react to 100 ng
of human MCP-1, -2, -3, and -4; MIP-1
; MIP-1
; or RANTES. All
experiments were performed at least in duplicate.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and PMA. Eotaxin mRNA expression
in U-937 cells (Fig.
1A)
was detectable 4 h after stimulation with TNF-
(10 ng/ml) and PMA
(0.1 µM) but not with LPS (1 µg/ml), IL-1
(10 ng/ml), IL-4 (10 ng/ml), or IFN-
(10 ng/ml). Treatment of U-937 cells with 100 ng
TNF-
/ml induced maximal eotaxin mRNA expression at 2 h, which
declined over the subsequent 6 h (Fig.
1B). In contrast, 0.1 µM PMA
induced maximal eotaxin mRNA expression 16 h after stimulation and
significant but declining expression in the subsequent 32 h (Fig.
1B). LPS (1 µg/ml), IL-1
(10 ng/ml), IL-4 (10 ng/ml), and IFN-
(10 ng/ml) did not induce
significant eotaxin mRNA expression at any of these time points (data
not shown). Neither did 4 h of incubation with LPS (0.01, 0.1, 1 and 10 µg/ml), IL-1
(0.1, 1, 10, and 100 ng/ml), IL-4 (0.1, 1, 10, and
100 ng/ml), or IFN-
(0.1, 1, 10, and 100 ng/ml) induce significant
eotaxin mRNA expression in this cell line (data not shown). The
following experiments were performed as a control for the effects of
these stimuli on monocytic cells. Biological effects of IFN-
(1-100 ng/ml) on IP-10 expression in U-937 cells were demonstrated
(see Fig. 3). IL-4 (10 ng/ml) decreased cytokine-induced eotaxin mRNA
expression, and LPS (1 µg/ml) and IL-1
(10 ng/ml) induced IL-8
expression in human monocytes (data not shown).
View larger version (29K):
[in a new window]
Fig. 1.
Northern blot analyses of eotaxin mRNA expression in U-937 cells.
A: effects of various stimuli on
eotaxin mRNA expression 4 h after stimulation.
B: time courses of eotaxin mRNA
expression after stimulation with tumor necrosis factor (TNF)- and
phorbol 12-myristate 13-acetate (PMA).
C: concentration (Conc)-response
effects of TNF-
and PMA on eotaxin mRNA expression. Cells were
harvested 2 h after stimulation with TNF-
and 16 h after stimulation
with PMA in these concentration-response studies. Each blot was
hybridized sequentially with eotaxin- and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH)-specific cDNA probes. A representative series of
blots is shown in A and
B (n = 3) and C (n = 2). LPS,
lipopolysaccharide; IL, interleukin; IFN, interferon; US,
unstimulated.
Concentration-response effects of TNF-
and PMA on eotaxin mRNA expression. The
concentration-response effects of TNF-
and PMA on eotaxin mRNA
expression are shown in Fig. 1C. The
addition of increasing doses from 0.1 to 10 ng/ml of TNF-
to the
medium was associated with a concentration-dependent increase in
eotaxin mRNA expression, with decreasing eotaxin expression observed at the highest concentration (100 ng/ml). Doses of 0.01-1 µM PMA induced significant eotaxin expression, with a striking elevation in
expression between 0.001 and 0.01 µM. Cells were harvested after 2 h
of stimulation with TNF-
and 16 h with PMA. RNA size markers, shown
in Fig. 1C, demonstrate a 0.8-kb
transcript of eotaxin gene and a 1.3-kb transcript of GAPDH gene.
Effects of dexamethasone and
IFN-. Pretreatment of U-937 cells with
increasing concentrations of dexamethasone was associated with a
concentration-dependent decrease in TNF-
(100 ng/ml)- and PMA (0.1 µM)-induced eotaxin mRNA expression (Fig.
2, A and B). Coincubation of U-937 cells with
1, 10, or 100 ng/ml of IFN-
had no significant effect on TNF-
-
and PMA-induced eotaxin mRNA expression, whereas IP-10 mRNA expression
was enhanced in the presence of IFN-
(Fig. 3,
A and
B). Our
IP-10-specific probe detected a single 1.3-kb band.
|
|
Effects of protein synthesis inhibitor
cycloheximide. TNF--induced eotaxin mRNA expression
was slightly decreased when protein synthesis was inhibited by 10 µg/ml of cycloheximide (Fig.
4A). In
contrast, PMA-induced eotaxin mRNA expression could not be detected in
the presence of cycloheximide (Fig.
4B). IP-10 mRNA expression was
slightly superinduced by cycloheximide with or without TNF-
or PMA
stimulation (data not shown).
|
TNF- concentrations in the culture
supernatant. TNF-
concentrations in the culture
supernatant of U-937 cells were less than 5 pg/ml 1, 2, 4, 8, 16, and
24 h after stimulation with PMA (0.1 µM). The concentration was 11.0 ± 1.0 pg/ml (mean ± SE; n = 4)
48 h after stimulation, which was less than the minimum exogenous concentration required to induce eotaxin expression as determined in
the concentration-response experiment.
Effects of TNF-
neutralization. PMA-induced eotaxin mRNA expression was
not significantly inhibited in the presence of anti-TNF-
neutralizing antibody (0.1 and 1 µg/ml) (Fig.
4C). The anti-TNF-
antibody (1 µg/ml) neutralized >90% of TNF-
effects on eotaxin mRNA
expression in U-937 cells when the TNF-
concentration was 1 ng/ml
and >70% when it was 10 ng/ml (data not shown).
Effects of protein kinase C inhibitor
staurosporine. Pretreatment of U-937 cells with
increasing concentrations of the PKC inhibitor staurosporine was
associated with a concentration-dependent increase in TNF--induced
eotaxin mRNA expression (Fig.
5A). In contrast, PMA-induced eotaxin expression was inhibited by staurosporine in a concentration-dependent manner (Fig.
5B). Staurosporine (50 nM) itself
did not induce eotaxin mRNA expression 2 and 16 h after its addition to
U-937 cell supernatant (data not shown).
|
Effects of nuclear factor-B inhibitor
DETC. Pretreatment of U-937 cells with 0.001-1 mM
of the nuclear factor-
B (NF-
B) inhibitor DETC was associated with
a concentration-dependent decrease in TNF-
-induced eotaxin mRNA
expression (Fig.
6A).
PMA-induced eotaxin expression was inhibited by 0.1-1 mM DETC, and
a steep decrease in eotaxin expression was observed between 0.01 and
0.1 mM (Fig. 6B).
|
NF-B binding activity in U-937
cells. Enhanced NF-
B binding
activity was demonstrated in U-937 cells after 2 h of stimulation with
TNF-
and 8 h of stimulation with PMA by EMSA (Fig.
6C). These results were consistent
with the kinetics of eotaxin mRNA expression induced by TNF-
and
PMA. Specific binding of the NF-
B consensus probe to the nuclear
extracts was suggested by the competition experiment with unlabeled
probe.
Immunocytochemical
staining. Minimal eotaxin
immunoreactivity was detectable in unstimulated U-937 cells (Fig.
7, A and
C). Compared with the unstimulated
cells, increased eotaxin immunoreactivity was observed in U-937 cells
after incubation with TNF- for 8 h and PMA for 48 h (Fig. 7,
B and
D). These observations were consistent with the eotaxin mRNA induction after stimulation with TNF-
or PMA.
|
Regulation of eotaxin mRNA expression in monocytes and
A549 cells. TNF- induced significant eotaxin mRNA
expression in monocytes and A549 cells, whereas PMA induced only faint
eotaxin expression in both cell types (Fig.
8, A and
B). TNF-
-induced eotaxin mRNA expression was enhanced by staurosporine and inhibited by DETC in
monocytes, which was similar to the findings in U-937 cells, although
the expression was diminished in the presence of cycloheximide in
monocytes (Fig. 8A). In contrast,
TNF-
-induced eotaxin mRNA expression was markedly enhanced in the
presence of cycloheximide in A549 cells. The expression was inhibited
by staurosporine and DETC in this cell line, which suggests that
TNF-
-induced eotaxin expression is partially mediated by PKC as well
as by NF-
B (Fig. 8B). PMA-induced
eotaxin mRNA expression was also markedly enhanced by cycloheximide in
A549 cells.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We demonstrated that PMA-induced eotaxin mRNA expression was of greater
magnitude and occurred later than TNF--induced eotaxin expression in
U-937 cells. Eotaxin protein was also detected by immunocytochemistry
in both TNF-
- and PMA-stimulated U-937 cells. The protein synthesis
inhibitor cycloheximide markedly diminished PMA-induced eotaxin mRNA
expression, although TNF-
-induced expression was not significantly
decreased by this reagent. The PKC inhibitor staurosporine inhibited
PMA-induced eotaxin mRNA expression but enhanced TNF-
-induced
expression. These results suggest that TNF-
and PMA augment eotaxin
expression in U-937 cells by distinct mechanisms.
Staurosporine is a well-described inhibitor of PKC, the activity of
which is related to its ability to bind the catalytic domain of PKC
(19). One of the important actions of PMA is its ability to activate
PKC. We demonstrated concentration-dependent inhibition of PMA-induced
eotaxin mRNA expression at moderate levels of staurosporine in U-937
cells. PMA-induced eotaxin expression therefore appears to be mediated
by PKC activation. When the cells were stimulated with TNF-, eotaxin
expression was enhanced in a concentration-dependent manner by similar
levels of staurosporine. We determined that staurosporine alone did not
significantly affect U-937 cell eotaxin mRNA expression, thus
suggesting that it increases TNF-
-induced eotaxin mRNA expression by
an indirect mechanism. One explanation for this increase relates to the
ability of staurosporine to increase the presence of TNF receptors on
the cell surface (33). These observations indicate that TNF-
can
induce eotaxin mRNA through PKC-independent pathways in U-937 cells.
TNF-
-induced eotaxin mRNA expression was also enhanced by
staurosporine in human monocytes, whereas the expression was inhibited
by this agent in the epithelial cell line A549. These results suggest that the TNF-
-triggered pathway may be regulated by PKC-independent mechanisms in human monocytes as well as in U-937 cells but that TNF-
can induce eotaxin mRNA expression at least partially through PKC-dependent mechanisms in epithelial cells.
An NF-B response element has been identified in the human eotaxin
promoter region near the transcription initiation site (11). EMSA
demonstrated enhanced NF-
B binding activity in U-937 cells 2 h after
stimulation with TNF-
and 8 h after stimulation with PMA. These
observations suggest that PMA- and TNF-
-induced eotaxin expression
is mediated by NF-
B activation. The mechanisms by which these
stimuli lead to NF-
B activation are complex, but PKC activation and
reactive oxygen intermediates are thought to be involved in these
pathways (20, 28). A previous study suggested that TNF-
can activate
NF-
B by PKC-independent mechanisms (20), which is consistent with
our finding that TNF-
-induced eotaxin mRNA expression does not
depend on PKC activation in U-937 cells. DETC is an NF-
B inhibitor
with antioxidative properties and is a chelator of heavy metals (15,
27). Its inhibitory effects on NF-
B activation are thought to be
related to the inhibition of the release of the inhibitor
I
B from the NF-
B-I
B complex in the cytoplasm
(12). DETC inhibited eotaxin mRNA expression in both PMA- and
TNF-
-stimulated U-937 cells at 0.1 and 1 mM, which is consistent
with our notion that NF-
B activation is a common element in the
mechanistic pathways triggered by PMA and TNF-
that induce eotaxin
expression in U-937 cells. These observations may be applicable to
human monocytes and epithelial cells because DETC also inhibited
TNF-
-induced eotaxin mRNA expression in these cell types.
We found that PMA-induced eotaxin mRNA expression was dependent on
protein synthesis, and it is known that PMA can increase TNF-
expression in U-937 cells (6). To determine whether PMA induced eotaxin
mRNA expression by TNF-
synthesis, we measured PMA-induced TNF-
protein levels in the culture supernatant and examined the effects of
anti-TNF-
neutralizing antibody on PMA-induced eotaxin mRNA
expression. The levels that we detected do not appear to be sufficient
to account for the magnitude of PMA-induced eotaxin mRNA expression
that we observed. Because TNF-
concentrations close to the cell
surface may have been higher than those we detected in cell
supernatants, we examined the effects of anti-TNF-
neutralizing antibody on PMA-induced eotaxin expression. The ability of this antibody to block TNF-
- but not PMA-induced eotaxin expression indicates that the PMA pathway is independent of TNF-
production in
U-937 cells. The dependence of this pathway on new protein synthesis
suggests that de novo synthesis of transcription factors or
RNase-repressive factors is involved in PMA-induced eotaxin mRNA
expression in this cell line. Similarly, TNF-
-induced eotaxin mRNA
expression was dependent on protein synthesis in human monocytes.
Although different mechanisms are involved in eotaxin mRNA expression
by TNF- and PMA in U-937 cells, inhibitory effects of dexamethasone
were observed in both pathways. Because NF-
B activation is thought
to be a common element, the effects of glucocorticoids could relate to
the well-described ability of the glucocorticoid receptor to inhibit
NF-
B binding to its response element (25). Alternatively,
dexamethasone may decrease the stability of eotaxin mRNA. Because the
ATTTA sequences were identified in the 3'-untranslated region of
the human eotaxin gene, glucocorticoids can promote the effects of
RNase, which degrades mRNA that contains AU-rich sequences (10, 23,
31). It is also possible that dexamethasone effects are mediated by the
human eotaxin promoter glucocorticoid response element (11). In any
case, part of the known eosinophil-suppressive effects of
glucocorticoids may relate to this ability to suppress eotaxin mRNA
expression.
TNF- induced eotaxin mRNA in human monocytes by protein
synthesis-dependent mechanisms. PMA induced prolonged and enhanced eotaxin expression in U-937 cells, which was also dependent on new
protein synthesis. In contrast, TNF-
- and PMA-induced eotaxin mRNA
was superinduced by cycloheximide in a human epithelial cell line,
A549. Cytokine-induced eotaxin mRNA expression, enhanced in the
presence of IFN-
in A549 cells, was thought to be mediated by
IFN-
response elements identified in the human eotaxin
promoter region (11, 17). However, these synergistic effects of IFN-
on eotaxin mRNA expression were not observed in U-937 cells despite the
findings that these cells could respond to IFN-
by increasing the
levels of IP-10 mRNA (30). These results suggest that human monocytic
cells can express eotaxin mRNA as well as epithelial cells, but the
expression is regulated differently in these two cell types.
Differential expression of eotaxin may be important for establishing
eotaxin gradients in tissue that allow it to function as an eosinophil
chemoattractant.
In summary, we have demonstrated that eotaxin expression is regulated
by different mechanisms in TNF-- and PMA-stimulated U-937 cells; the
PMA-triggered pathway is mediated by de novo protein synthesis and PKC
activation, whereas the TNF-
-triggered pathway does not depend on
either of these steps. However, NF-
B activation is thought to be a
common downstream element in these two distinct cascades and may also
contribute to eotaxin mRNA induction in human monocytes and epithelial
cells, as suggested by the response to the NF-
B inhibitor DETC.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Jeffrey M. Drazen for his support.
![]() |
FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grant HL-03283 and National Institute of Allergy and Infectious Diseases Grant AI-40618. A. D. Luster is a recipient of a Cancer Research Institute (Benjamin Scholar Funding) Investigator Award.
Address for reprint requests: C. M. Lilly, Respiratory Division, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.
Received 24 July 1997; accepted in final form 1 May 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, C. L.,
and
H. L. Spiegelberg.
Macrophage receptors for IgE: binding of IgE to specific IgE Fc receptors on a human macrophage cell line, U-937.
J. Immunol.
126:
2470-2473,
1981
2.
Baumgartner, R. A.,
V. A. Deramo,
and
M. A. Beaven.
Constitutive and inducible mechanisms for synthesis and release of cytokines in immune cell lines.
J. Immunol.
157:
4087-4093,
1996[Abstract].
3.
Bentley, A. M.,
Q. Hamid,
D. S. Robinson,
E. Schotman,
Q. Meng,
B. Assoufi,
A. B. Kay,
and
S. R. Durham.
Prednisolone treatment in asthma: reduction in the numbers of eosinophils, T cells, tryptase-only positive mast cells, and modulation of IL-4, IL-5, and interferon-gamma cytokine gene expression within the bronchial mucosa.
Am. J. Respir. Crit. Care Med.
153:
551-556,
1996[Abstract].
4.
Borish, L.,
J. J. Mascali,
J. Dishuck,
W. R. Beam,
R. J. Martin,
and
L. J. Rosenwasser.
Detection of alveolar macrophage-derived IL-1 in asthma: inhibition with corticosteroids.
J. Immunol.
149:
3078-3082,
1992
5.
Brieland, J. K.,
M. L. Jones,
C. M. Flory,
G. R. Miller,
J. S. Warren,
S. H. Phan,
and
J. C. Fantone.
Expression of monocyte chemoattractant protein-1 (MCP-1) by rat alveolar macrophages during chronic lung injury.
Am. J. Respir. Cell Mol. Biol.
9:
300-305,
1993[Medline].
6.
Cannistra, S. A.,
A. Rambaldi,
D. R. Spriggs,
F. Herrmann,
D. Kufe,
and
J. D. Griffin.
Human granulocyte-macrophage colony-stimulating factor induces expression of the tumor necrosis factor gene by the U-937 cell line and by normal human monocytes.
J. Clin. Invest.
79:
1720-1728,
1987[Medline].
7.
Collins, P. D.,
S. Marleau,
D. A. Griffiths-Johnson,
P. J. Jose,
and
T. J. Williams.
Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo.
J. Exp. Med.
182:
1169-1174,
1995[Abstract].
8.
Daugherty, B. L.,
S. J. Siciliano,
J. A. DeMartino,
L. Malkowitz,
A. Sirotina,
and
M. S. Springer.
Cloning, expression, and characterization of the human eosinophil eotaxin receptor.
J. Exp. Med.
183:
2349-2354,
1996[Abstract].
9.
Garcia-Zepeda, E. A.,
C. Combadiere,
M. E. Rothenberg,
M. N. Sarafi,
F. Lavigne,
Q. Hamid,
P. M. Murphy,
and
A. D. Luster.
Human monocyte chemoattractant protein (MCP)-4 is a novel CC chemokine with activities on monocytes, eosinophils, and basophils induced in allergic and nonallergic inflammation that signals through the CC chemokine receptors (CCR)-2 and -3.
J. Immunol.
157:
5613-5626,
1996[Abstract].
10.
Garcia-Zepeda, E. A.,
M. E. Rothenberg,
R. T. Ownbey,
J. Celestin,
P. Leder,
and
A. D. Luster.
Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia.
Nat. Med.
2:
449-456,
1996[Medline].
11.
Garcia-Zepeda, E. A.,
M. E. Rothenberg,
S. Weremowicz,
M. N. Sarafi,
C. C. Morton,
and
A. D. Luster.
Genomic organization, complete sequence, and chromosomal location of the gene for human eotaxin (SCYA11), an eosinophil-specific CC chemokine.
Genomics
41:
471-476,
1997[Medline].
12.
Henkel, T.,
T. Machleidt,
I. Alkalay,
M. Krönke,
Y. Ben-Neriah,
and
P. A. Baeuerle.
Rapid proteolysis of IB-
is necessary for activation of transcription factor NF-
B.
Nature
365:
182-185,
1993[Medline].
13.
Kita, H.,
and
G. J. Gleich.
Chemokines active on eosinophils: potential roles in allergic inflammation.
J. Exp. Med.
183:
2421-2426,
1996[Medline].
14.
Kitaura, M.,
T. Nakajima,
T. Imai,
S. Harada,
C. Combadiere,
H. L. Tiffany,
P. M. Murphy,
and
O. Yoshie.
Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3.
J. Biol. Chem.
271:
7725-7730,
1996
15.
Kwon, G.,
J. A. Corbett,
C. P. Rodi,
P. Sullivan,
and
M. L. McDaniel.
Interleukin-1-induced nitric oxide synthase expression by rat pancreatic
-cells: evidence for the involvement of nuclear factor
B in the signaling mechanism.
Endocrinology
136:
4790-4795,
1995[Abstract].
16.
Lamkhioued, B.,
P. M. Renzi,
S. Abi-Younes,
E. A. Garcia-Zepeda,
Z. Allakhverdi,
O. Ghaffar,
M. D. Rothenberg,
A. D. Luster,
and
Q. Hamid.
Increased expression of eotaxin in bronchoalveolar lavage and airways of asthmatics contributes to the chemotaxis of eosinophils to the site of inflammation.
J. Immunol.
159:
4593-4601,
1997[Abstract].
17.
Lilly, C. M.,
H. Nakamura,
H. Kesselman,
C. Nagler-Anderson,
K. Asano,
E. A. Garcia-Zepeda,
M. E. Rothenberg,
J. M. Drazen,
and
A. D. Luster.
Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids.
J. Clin. Invest.
99:
1767-1773,
1997
18.
Luster, A. D.,
J. C. Unkeless,
and
J. V. Ravetch.
-Interferon transcriptionally regulates an early-response gene containing homology to platelet proteins.
Nature
315:
672-676,
1985[Medline].
19.
Makishima, M.,
Y. Honma,
M. Hozumi,
K. Sampi,
K. Motoyoshi,
N. Nagata,
and
M. Hattori.
Differentiation of human myeloblastic leukemia ML-1 cells into macrophages by staurosporine, an inhibitor of protein kinase activities.
Exp. Hematol.
21:
839-845,
1993[Medline].
20.
Meichle, A.,
S. Schütze,
G. Hensel,
D. Brunsing,
and
M. Krönke.
Protein kinase C-independent activation of nuclear factor B by tumor necrosis factor.
J. Biol. Chem.
265:
8339-8343,
1990
21.
Nakamura, H.,
S. Fujishima,
Y. Waki,
T. Urano,
K. Sayama,
F. Sakamaki,
T. Terashima,
K. Soejima,
S. Tasaka,
A. Ishizaka,
T. Kawashiro,
and
M. Kanazawa.
Priming of alveolar macrophages for interleukin-8 production in patients with idiopathic pulmonary fibrosis.
Am. J. Respir. Crit. Care Med.
152:
1579-1586,
1995[Abstract].
22.
Navarro, S.,
N. Debili,
J. F. Bernaudin,
W. Vainchenker,
and
J. Doly.
Regulation of the expression of IL-6 in human monocytes.
J. Immunol.
142:
4339-4345,
1989
23.
Peppel, K.,
J. M. Vinci,
and
C. Baglioni.
The AU-rich sequences in the 3' untranslated region mediate the increased turnover of interferon mRNA induced by glucocorticoids.
J. Exp. Med.
173:
349-355,
1991[Abstract].
24.
Ponath, P. D.,
S. Qin,
D. J. Ringler,
I. Clark-Lewis,
J. Wang,
N. Kassam,
H. Smith,
X. Shi,
J. A. Gonzalo,
W. Newman,
J. C. Gutierrez-Ramos,
and
C. R. Mackay.
Cloning of the human eosinophil chemoattractant, eotaxin: expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils.
J. Clin. Invest.
97:
604-612,
1996
25.
Ray, A.,
and
K. E. Prefontaine.
Physical association and functional antagonism between the p65 subunit of transcription factor NF-B and the glucocorticoid receptor.
Proc. Natl. Acad. Sci. USA
91:
752-756,
1994[Abstract].
26.
Resnick, M. B.,
and
P. F. Weller.
Mechanisms of eosinophil recruitment.
Am. J. Respir. Cell Mol. Biol.
8:
349-355,
1993[Medline].
27.
Schreck, R.,
B. Meier,
D. N. Männel,
W. Dröge,
and
P. A. Baeuerle.
Dithiocarbamates as potent inhibitors of nuclear factor B activation in intact cells.
J. Exp. Med.
175:
1181-1194,
1992[Abstract].
28.
Schreck, R.,
P. Rieber,
and
P. A. Baeuerle.
Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-B transcription factor and HIV-1.
EMBO J.
10:
2247-2258,
1991[Abstract].
29.
Takeshita, S.,
J. R. Gage,
T. Kishimoto,
D. L. Vredevoe,
and
O. Martínez-Maza.
Differential regulation of IL-6 gene transcription and expression by IL-4 and IL-10 in human monocytic cell lines.
J. Immunol.
156:
2591-2598,
1996[Abstract].
30.
Taub, D. D.,
A. R. Lloyd,
K. Conlon,
J. M. Wang,
J. R. Ortaldo,
A. Harada,
K. Matsushima,
D. J. Kelvin,
and
J. J. Oppenheim.
Recombinant human interferon-inducible protein 10 is a chemoattractant for human monocytes and T lymphocytes and promotes T cell adhesion to endothelial cells.
J. Exp. Med.
177:
1809-1814,
1993[Abstract].
31.
Tobler, A.,
R. Meier,
M. Seitz,
B. Dewald,
M. Baggiolini,
and
M. F. Fey.
Glucocorticoids downregulate gene expression of GM-CSF, NAP-1/IL-8, and IL-6, but not of M-CSF in human fibroblasts.
Blood
79:
45-51,
1992[Abstract].
32.
Weller, P. F.
The immunobiology of eosinophils.
N. Engl. J. Med.
324:
1110-1118,
1991[Medline].
33.
Zhang, L.,
M. Higuchi,
K. Totpal,
M. M. Chaturvedi,
and
B. B. Aggarwal.
Staurosporine induces the cell surface expression of both forms of human tumor necrosis factor receptors on myeloid and epithelial cells and modulates ligand-induced cellular response.
J. Biol. Chem.
269:
10270-10279,
1994