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Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 558-566
Shiga Toxin Type 1 Activates Tumor Necrosis Factor- Gene
Transcription and Nuclear Translocation of the Transcriptional
Activators Nuclear Factor- B and Activator Protein-1
By
Ramesh Sakiri,
Belakere Ramegowda, and
Vernon L. Tesh
From the Department of Medical Microbiology and Immunology, Texas A&M
University Health Science Center, College Station, TX.
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ABSTRACT |
Shiga toxins (Stxs) produced by Shigella dysenteriae 1 and
Escherichia coli have been implicated in the pathogenesis of
bloody diarrhea, acute renal failure, and neurologic abnormalities. The pathologic hallmark of Stx-mediated tissue damage is the development of
vascular lesions in which endothelial cells are swollen and detached
from underlying basement membranes. However, in vitro studies using
human vascular endothelial cells demonstrated minimal Stx-induced
cytopathic effects, unless the target cells were also incubated with
the proinflammatory cytokines tumor necrosis factor- (TNF-) or
interleukin-1 (IL-1). These cytokines have been shown to
upregulate the expression of the Stx-binding membrane glycolipid globotriaosylceramide (Gb3). We show here that purified
Stx1 induces TNF secretion by a human monocytic cell line, THP-1, in a
dose- and time-dependent manner. Treatment of cells with both
lipopolysaccharides (LPS) and Stx1 results in augmented TNF production.
Treatment with the nontoxic Gb3-binding subunit of Stx1 or
with an anti-Gb3 monoclonal antibody did not trigger TNF
production. Northern blot analyses show that Stx1 causes increased
TNF- production through transcriptional activation. Increased levels
of TNF- mRNA are preceded by the nuclear translocation of the
transcriptional activators NF- B and AP-1 and the loss of cytoplasmic
IB-. These data are the first to show that, in addition to direct
cytotoxicity, Stxs possess cellular signaling capabilities sufficient
to induce the synthesis of cytokines that may be necessary for target
cell sensitization and the development of vascular lesions.
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INTRODUCTION |
THE SHIGA TOXIN family of bacterial
protein toxins consists of Shiga toxin (Stx), produced by Shigella
dysenteriae serotype 1, and a group of closely related toxins
designated Stx1, 2, 2c, and 2e produced by Escherichia coli.
All members of the Stx family are AB5 holotoxins,
consisting of a single enzymatic A-subunit in noncovalent association
with a pentamer of identical B-subunits. Stx binding to susceptible
cells is mediated by B-subunit interaction with the neutral glycolipid
globotriaosylceramide (Gb3).1,2 The toxins are
internalized by a receptor-mediated endocytic mechanism and undergo
retrograde translocation through the Golgi stacks to the endoplasmic
reticulum, where a fragment of the A-subunit may traverse the ER
membrane and associate with ribosomes.3 The A-subunit
cleaves a single adenine residue located in a prominent loop structure
of the 28 S rRNA component of eukaryotic ribosomes, and depurination
results in protein synthesis inhibition.4,5
Stxs may cause disease in humans by damaging intestinal, renal, and
central nervous system capillary blood vessels, resulting in an
exacerbation of colonic ulceration and bloody diarrhea and the
development of acute renal failure, seizures, and death.6 Glomerular vascular lesions caused by Stxs are characterized by endothelial cell swelling and detachment from glomerular basement membranes and the deposition of micro-thrombi within glomeruli. However, a direct cytotoxic effect of purified Stxs on human vascular endothelial cells in vitro was minimal, unless the target cells were
cultured in the presence of the proinflammatory cytokines tumor
necrosis factor- (TNF-) or interleukin-1
(IL-1).7,8 Subsequently, van der Kar et al9
showed that TNF- acts on human endothelial cells to upregulate the
expression of the toxin-binding glycolipid Gb3, suggesting
that the host response to Stxs may participate in the development of
vascular damage. We found that human vascular endothelial cells do not
synthesize TNF- or IL-1 when treated with purified Stxs in vitro,
suggesting that other cell types may be necessary to produce the
cytokines involved in glycolipid modulation on target cells. Murine
peritoneal macrophages, human peripheral blood monocytes, and human
monocytic cell lines are relatively resistant to the cytotoxic action
of Stxs, express low levels of membrane Gb3, and respond to
toxin stimulation by secreting TNF-, IL-1, and
IL-6.10-12 However, the mechanism by which Stxs mediate
cytokine induction is not known.
TNF- gene expression is tightly controlled, including regulation of
transcription initiation, mRNA processivity, and translational and
posttranslational regulatory controls. In the experiments reported
here, we investigated Stx1-mediated human monocyte TNF protein
production, TNF- mRNA induction, and the cellular signaling events
that may be involved in cytokine induction. We focused our studies on
the transcriptional activators nuclear factor-B (NF-B) and
activator protein-1 (AP-1), protein complexes that have been shown to
translocate to the nucleus and upregulate transcription in response to
a variety of stimuli.13,14
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MATERIALS AND METHODS |
Cells.
Human peripheral blood monocytes (PBMn) were derived from blood
collected from healthy volunteers. Mononuclear cells were separated by
Histopaque 1077 (Sigma, St Louis, MO) gradient
centrifugation, and plastic nonadherent cells were removed after 1 hour
of incubation at 37°C. The human myelogenous leukemia cell line
THP-115 was purchased from ATCC (Rockville, MD). All
monocytic cells were maintained in RPMI-1640 (GIBCO-BRL, Grand Island,
NY) supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), amphotericin B (2.0 µg/mL), and 10% fetal bovine serum
(FBS; Hyclone Laboratories, Logan, UT) at 37°C in humidified 5%
CO2. Differentiated THP-1 cells have been shown to share
many of the physiological functions of primary monocyte-derived
macrophages.16 In all the experiments reported here, THP-1
cells (1 × 106 cells/mL) were induced to
differentiate to the mature macrophage-like state by treatment with
12-0-tetradecanoylphorbol-13-acetate (TPA; Sigma) at 50 ng/mL
for 48 hours in 100-mm culture dishes. Differentiated, plastic-adherent
cells were washed twice with cold Dulbecco's phosphate-buffered saline
(PBS; GIBCO-BRL) and incubated with fresh medium lacking TPA for 3 to 4 days with daily medium changes before use in assays. Although TPA
treatment of THP-1 cells resulted in transient increases in TNF
production and NF-B nuclear translocation, these activities returned
to baseline (unstimulated) levels 3 to 4 days after TPA treatment (data
not shown). The murine fibroblast cell line L929 was maintained in
Iscove's modified Dulbecco's medium (IMDM; Celox Corp, Hopkins, MN)
containing 5% FBS at 37°C in humidified 5% CO2.
Toxins and cytokine inducers.
Purified Stx1 was prepared as previously described.10
Before use, toxin preparations were passed through ActiClean Etox columns (Sterogene Bioseparations, Arcadia, CA) to remove traces of
endotoxin contaminants. Purified pentameric Stx1 B-subunits were the
kind gift of Dr David Acheson (Tufts University School of Medicine,
Boston, MA). Purified lipopolysaccharides (LPS) derived from
Escherichia coli 0111:B4 were purchased from Sigma. Murine monoclonal IgM antibody pK002, directed against Gb3, was
purchased from Accurate Chemical Corp (Westbury, NY).
TNF bioactivity assay.
TNF bioactivity in THP-1 supernatants from untreated control cells and
cells treated with Stx1 and/or LPS was determined by the lysis
of actinomycin-D (act-D; Sigma) -treated L929 murine fibroblasts as
previously described.10 Briefly, L929 cells were cultured
in 96-well microtiter plates at a density of 2 × 105
cells/mL in IMDM supplemented with 5% FBS at 37°C in 5%
humidified CO2. Dilutions of macrophage supernatants or
recombinant human TNF- (R&D Systems, Minneapolis, MN) were made in
IMDM such that the final act-D concentration was 1.0 µg/mL. The
dilutions were added to 5 replicate wells of L929 cells in microtiter
plates and incubated for 18 hours. Twenty-five microliters of a 5.0 mg/mL stock solution of
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT;
Sigma) was added to each well and incubated at 37°C for 2 hours.
The cells were lysed and the formazan dye was extracted from the cells
with 50% N,N-dimethylformamide and 20% sodium dodecyl sulfate
(SDS). A570 was measured (Dynatech MR5000; Dynatech
Laboratories, Chantilly, VA) and L929 survival values were determined.
TNF bioactivity in the macrophage supernatants was calculated by
substituting the corresponding sample values in linear regression
equations generated by the recombinant human TNF- standard curve.
Direct treatment of L929 cells with act-D and Stx1 consistently
resulted in less than 10% cytotoxicity compared with untreated cells.
Statistical analyses using Student's paired t-test were
performed with Microsoft Excel version 5.0 software (Microsoft Corp,
Redmond, WA).
Isolation and analysis of total cellular RNA.
Total cellular RNA was isolated by the acid guanidinium isothiocyanate
extraction method17 using Ultraspec II RNA isolation kits
(Biotecx Laboratories, Houston, TX). RNA purity was assessed by
OD260/OD280 readings. Ten micrograms of RNA per
lane was electrophoresed through 0.8% agarose-2 mol/L formaldehyde
gels in 1× MOPS running buffer at 50 V for 2 to 3 hours. RNA was
then transferred onto positively charged nylon membranes (GeneScreen
Plus; NEN Dupont, Boston, MA) using the Turboblotter Rapid Downward
Transfer System (Schleicher & Schuell, Keene, NH) and cross-linked by
exposing the membrane to UV light (254 nm) for a total dose of 120 mJ/cm2 using a UV cross-linker (Bio-Rad, Hercules, CA). A
human TNF- probe (5 -ATCTCTCAGCTCCACGCCATTGGCCAGGAG-3;
Clonetech, Palo Alto, CA) was end-labeled with
[ 32P]-ATP as per the manufacturer's instructions. 18S
RNA antisense control template (Ambion, Inc, Austin, TX) was random
prime labeled using Megaprime DNA labeling kits (Amersham Corp,
Arlington Heights, IL). After labeling, unincorporated nucleotides were
removed using G-25 Sephadex columns (5 prime 3 prime, Inc, Boulder,
CO). The membranes were prehybridized for 15 minutes at 42°C for
the human TNF- probe or 65°C for the 18S RNA probe in 7 to 10 mL
of Rapid-hyb Hybridization Buffer (Amersham). After prehybridization,
106 cpm TNF- probe per milliliter of hybridization
buffer was added and the hybridization was performed for 2 to 4 hours
at 42°C. The membranes were washed in 2× SSC, 0.1% SDS for
10 minutes at room temperature and exposed to a phosphoimager screen
overnight. The screen was analyzed on a phosphoimager (Molecular
Dynamics, Sunnyvale, CA) and the sums of counts above background were
calculated using ImageQuant software (Molecular Dynamics). The
membranes were stripped by boiling in 0.1× SSC/0.1% SDS twice
for 15 minutes and reprobed at 65°C with 18S antisense RNA as an
internal control of RNA loading. Ratios of counts of TNF- mRNA:18S
RNA were calculated. To minimize interassay variability, relative
levels of TNF- mRNA from three separate experiments are shown by
expressing the ratios as percentages above basal levels using the
formula: ([stimulated ratio of counts TNF-:18S]  [unstimulated ratio of counts TNF:18S])/(unstimulated ratio of
counts TNF:18S) × 100.
DNA-protein interaction and electrophoretic mobility shift assay.
TPA-differentiated THP-1 cells (1 × 106/mL) were
stimulated with different concentrations of Stx1 (10 to 800 ng/mL) for
dose-response experiments and with 400 ng/mL of Stx1 for kinetics
studies. The cells were washed twice with cold PBS before nuclear
extract preparation. Nuclear extracts were prepared according to the
methods of Dignam et al18 and Schreiber et
al.19 Protein concentrations of nuclear extracts were
determined by the Bradford method (Pierce Chemical Co, Rockford,
IL20). A NF-B binding site-specific
oligonucleotide containing two tandemly repeated HIV-1 long terminal
repeat enhancers
(5-ATCAGGGACTTTCCGCTGGGGACTTTCCG-321) and a
second mutant oligonucleotide lacking the NF-B binding sites
(5-AGGATGGGAGTGTGATATATCCTTGAT-3) were synthesized. An AP-1 consensus sequence oligonucleotide containing a binding site for
C-Jun homodimer and Jun/Fos heterodimer complexes
(5-CGCTTGATGACTCAGCCGGAA-3) and a corresponding AP-1
oligonucleotide with a CA to TG substitution in the AP-1 binding motif
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All
oligonucleotides were end-labeled with [32P]-ATP using T4
polynucleotide kinase and unincorporated [32P]-ATP was
removed with Sephadex G-25 spin columns. DNA-protein interactions were
performed by incubating 5.0 µg of nuclear extract with 20,000 cpm of
[32P]-end labeled double-stranded NF-B site-specific
or AP-1 site-specific probe in the presence of 1.0 to 2.0 µg of poly
[dI-dC] (Pharmacia, Piscataway, NJ) in binding buffer (10 mmol/L Tris
HCl [pH 5], 10% glycerol, 1.0 mmol/L EDTA, 40 mmol/L KCl, 1.0 mmol/L
dithiothreitol, and 4.0 mmol/L MgCl2) for 30 minutes at
room temperature. In some experiments, nuclear extracts were incubated
with 25 or 50 molar excess of unlabeled probe or with end-labeled
mutated oligonucleotide with substitutions in binding motifs to examine
the specificity of NF-B or AP-1 binding to the DNA. After
incubation, samples were loaded onto nondenaturing 4% polyacrylamide
gels (acrylamide:bis-acrylamide 30:1 [wt:wt]) and electrophoresis was
performed using 0.25× Tris-borate running buffer (0.089 mol/L
Tris-borate, 0.089 mol/L boric acid, and 0.002 mol/L EDTA [pH 8]) at
180 V for 1 to 2 hours at 4°C. The gels were dried, radiolabeled
bands were visualized by phosphoimager, and counts were analyzed using
ImageQuant sofware.
Super-shift assays.
To identify specific NF-B proteins involved in DNA binding, 5.0 µg
of nuclear extracts was incubated with 1.0 µL of rabbit antisera
directed against human p50, p65, Rel B, or c-Rel proteins (kind gift of
Dr Nancy R. Rice, National Cancer Institute, Frederick Cancer Research
and Development Center, Frederick, MD22) for 10 minutes at
room temperature. After incubation, labeled NF-B specific probe was
added and the reaction was allowed to proceed for 30 minutes at room
temperature before electrophoretic mobility shift assays were
performed. Specificity of super-shifts was tested by adding 25 to 50 molar excess of the NF-B binding site-specific double-stranded
unlabeled oligonucleotide before incubation with the labeled probe.
Western immunoblots for IB-.
Western immunoblot analyses were performed using 5.0 µg of
cytoplasmic extracts prepared from Stx1-treated cells by the method of
Dignam et al.18 Proteins were resolved in 10% tris-glycine gels and electrotransferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin in PBS/0.1% Tween and probed with rabbit polyclonal anti-IB- antisera (kind gift of Dr
Nancy Rice23). Primary antibody binding was detected by
using peroxidase-conjugated antirabbit Ig antibodies. Blots were
developed by the addition of peroxidase substrate with enhancement by
ECL solution (Amersham) and exposed to blue sensitive films (Midwest Scientific, St Louis, MO). Bands were quantitated by densitometric scanning using Alpha-imager 3.21 software (Innotech, San Leandro, CA).
Correlations between NF-B translocation and I-B degradation were
evaluated by the Pearson product moment correlation test.
| |
RESULTS |
Dose response and kinetics of TNF production by Stx1 treated THP-1
cells.
We previously demonstrated that human PBMn and differentiated THP-1
cells were relatively insensitive to the cytotoxic action of purified
Stxs, expressed low levels of Gb3, and respond to Stxs by
secreting proinflammatory cytokines.11 The dose response of
TNF production by differentiated THP-1 cells incubated with serial
dilutions of purified Stx1 is depicted in
Fig 1. Stx1 appeared to be a less potent
inducer of TNF production in comparison to LPS, because treatment of
THP-1 cells with 800 ng/mL Stx1 generated approximately 75% of the
soluble TNF bioactivity stimulated by 200 ng/mL of LPS. The holotoxin
molecule was necessary to induce TNF secretion, because Gb3
binding by purified Stx1 B-subunits (400 ng/mL) or by an
anti-Gb3 monoclonal antibody (10 µg/mL) did not trigger
TNF synthesis and secretion above basal levels (~25 pg/mL). Northern
blot analyses showed that the dose-dependent production of soluble TNF
correlated with increased levels of TNF- mRNA transcripts isolated
from toxin-treated cells (Fig 2). Thus,
increased TNF production stimulated by Stx1 is mediated, at least in
part, at the transcriptional level.
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| Fig 1.
Dose response of TNF production by THP-1 cells stimulated
with Stx1. Differentiated THP-1 cells were incubated with the indicated concentrations of Stx1 or LPS for 12 hours. Cell-free culture supernatants were collected and TNF bioactivity was quantitated by L929
assay as outlined in the Materials and Methods. Data are expressed as
the mean (in picograms per milliliter) ± SEM of three separate
experiments.
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| Fig 2.
Dose response of TNF- mRNA production by THP-1 cells
stimulated with Stx1. (A) Differentiated THP-1 cells were incubated with the indicated concentrations of Stx1 for 12 hours. The cells were
lysed and total RNA was extracted. Northern blot analysis was performed
to determine the levels of TNF- mRNA and 18S RNA. (B) The values
shown are the ratios of cpm TNF- mRNA:cpm 18 S RNA of one
representative blot from three separate experiments. All test values
are statistically different from unstimulated cells by Student's
paired t-test (P < .1).
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The kinetics of TNF production were examined by incubating THP-1 cells
with purified Stx1, LPS, or both for varying timepoints and then
measuring TNF bioactivity in macrophage supernatants using the L929
cytotoxicity assay as described in the Materials and Methods. Treatment
of THP-1 cells with Stx1 or LPS alone resulted in the rapid induction
of TNF expression, with bioactivity peaking between 3 and 6 hours
(Table 1). Stx1- and LPS-induced TNF
activity then decreases to 72% to 77% of peak values over the last 6 hours of the experiment. Treatment of THP-1 cells with the combination of Stx1 and LPS resulted in synergistically increased induction of
soluble TNF bioactivity compared with treatment with either stimulant
alone. However, the kinetics of TNF protein production were similar to
that of Stx1 or LPS alone (Table 1).
Northern blot analyses were performed to correlate the kinetics of
TNF- gene transcriptional activation with the appearance of TNF
bioactivity in macrophage supernatants. Maximal TNF- mRNA levels
after Stx1 or LPS treatment of THP-1 cells preceded maximal soluble TNF
bioactivity, peaking at 2 hours and 0.5 hours, respectively (Fig 3). Whereas LPS stimulated a transient
increase in TNF- transcripts, with mRNA levels returning to basal
values within 3 hours, Stx1 stimulated a prolonged elevation of TNF-
mRNA. When both Stx1 and LPS were used as stimulants, peak levels of TNF- mRNA were detected at 2 hours, and the levels of induced transcripts were higher than the levels induced by Stx1 or LPS alone.
The kinetics of LPS + Stx1 mediated TNF- mRNA induction more closely
resembled that induced by treatment of THP-1 cells with Stx1 alone in
that mRNA levels remained elevated over the course of the experiment.
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| Fig 3.
Kinetics of TNF- mRNA induction in THP-1 cells
stimulated with Stx1. Differentiated THP-1 cells were incubated with
400 ng/mL of Stx1, 200 ng/mL of LPS, or a combination of both for the
time points indicated. The cells were lysed and total RNA was
extracted. Northern blot analysis was performed to determine the levels
of TNF- mRNA and 18 S RNA. The ratios of cpm TNF- mRNA:cpm 18 S RNA were calculated as detailed in the Materials and Methods. The data
shown are the mean cpm ± SEM from three separate experiments.
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Dose response of NF-B activation by Stx1.
To elucidate the mechanism(s) by which Stx1, a potent protein synthesis
inhibitor in toxin-sensitive cells, induces the transcriptional activation of the TNF- gene in PBMns and THP-1 cells, we measured the nuclear translocation of two transcriptional activator complexes, NF-B and AP-1, in response to toxin treatment. Treatment of
macrophages with LPS or pharmacologic agents, such as phorbol diesters,
is known to induce NF-B and AP-1 nuclear translocation, and both NF-B and AP-1 are involved in the regulation of many genes encoding cytokines, cytokine receptors, and acute-phase proteins involved in
inflammatory responses.13,14 PBMns and differentiated THP-1 cells were incubated with increasing concentrations of Stx1 for 2 hours. Nuclear extracts were prepared and incubated with a radiolabeled oligonucleotide containing two tandem NF-B binding
sites.21 Translocation of NF-B from the cytosol to the
nucleus after toxin exposure was assessed by electrophoretic mobility
shift in nondenaturing polyacrylamide gels associated with the binding
of NF-B to the radiolabeled target DNA as outlined in the Materials
and Methods. Nuclear extracts prepared from untreated control cells
contained low basal levels of NF-B binding activity
(Fig 4). However, upon Stx1 treatment,
THP-1 cells (Fig 4A) and PBMn (Fig 4B) displayed concentration-dependent increased NF-B binding activity. Stx1 concentrations as low as 100 ng/mL induced detectable NF-B binding activity, which reached a maximum at 800 ng/mL Stx1. A control oligonucleotide containing mutated NF-B binding sites and
competition with excess unlabeled NF-B-specific oligonucleotide were
used to determine the specificity of DNA binding.
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| Fig 4.
Stx1 dose response of NF-B nuclear translocation. (A)
Differentiated THP-1 cells or (B) human PBMn were incubated with
varying concentrations of Stx1 for 2 hours. Nuclear extracts were
prepared and electrophoretic mobility shift assays were performed in
the presence of a [32P]-labeled double-stranded
NF-B-binding oligonucleotide or an oligonucleotide containing a
substitution in the NF-B binding motif. The data shown in (C) are
the ratios of counts in Stx1-treated cells per counts in control cells
(basal) ± SEM from three separate experiments.
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Kinetics of Stx1 stimulated NF-B translocation and
IB- degradation.
Earlier studies have shown that the translocation of an NF-B complex
to the nuclei of LPS-treated THP-1 cells occurs within 30 minutes.24 To investigate the time course of nuclear
localization of NF-B in response to Stx1, TPA-differentiated THP-1
cells were stimulated with 400 ng/mL of Stx1 for various time points
and nuclear extracts analyzed for NF-B binding. In accordance with the kinetics of Stx1-mediated soluble TNF activity and TNF- mRNA induction, inducible NF-B binding activity was detectable at 60 minutes and was maximal by 120 minutes (Fig
5A and C). The response began to decrease by 3 hours after Stx1
treatment; however, the level of NF-B induction remained elevated
compared with basal values. Cold oligonucleotide competition and mutant
oligonucleotide treatment demonstrated that NF-B binding activity
was specific. The kinetics of the loss of immunoreactive IB- in
the cytoplasm were directly related to the kinetics of
NF-B nuclear translocation (r = .98, P  .01; Fig 5B and C).
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| Fig 5.
Kinetics of Stx1-induced nuclear translocation of NF-B
complexes and IB- degradation. Differentiated THP-1 cells were
incubated with 400 ng/mL of Stx1 for the indicated times. (A) Nuclear
extracts were prepared and electrophoretic mobility shift assays were
performed in the presence of a [32P]-labeled
double-stranded NF-B binding oligonucleotide. Competition assays
were performed by incubating nuclear extracts with a 25 molar excess of
unlabeled oligonucleotide. Specificity of binding was assessed by
incubating nuclear extracts with a radiolabeled oligonucleotide
containing a substitution in the NF-B binding motif. (B) Western
blot of IB- degradation in the cytoplasm. TNF- treatment of
THP-1 cells for 30 minutes was a positive control for IB-
degradation. (C) Mean NF-B binding activity (cpm ± SEM) and
IB- reactivity (densitometric units) from six and three separate
experiments, respectively.
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The NF-B complex translocated by Stx1 treatment is
composed of p50 and p65 heterodimers.
The transcriptional activator complex NF-B may be composed of
homodimeric or heterodimeric proteins designated p50, p65 (relA), c-rel, relB, and p52. All members of the NF-B/rel family share an
approximately 300 amino acid region of homology with the c-Rel protooncogene that is essential for complex dimerization and nuclear translocation.13 To assess the composition of the nuclear
factors translocating to nuclei and binding NF-B sites in response
to Stx1 treatment of THP-1 cells, a panel of rabbit antibodies against specific proteins of the human NF-B/Rel family was used in
electrophoretic mobility shift assays.22 Nuclear extracts
incubated with p50 and p65 antisera displayed an altered
electrophoretic mobility pattern (supershift) consistent with the
binding of the antisera to specific NF-B proteins
(Fig 6). No shifts in electrophoretic mobility were observed with either relB or C-rel antisera (data not
shown). Increasing the concentration of anti-p50 or anti-p65 antibodies
did not change the supershift pattern. Supershifts observed with p50
and p65 antisera were specific, because the patterns could be
eliminated by treatment with excess unlabeled oligonucleotide (Fig 6,
lanes 4 and 8).
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| Fig 6.
Stx1-induced NF-B complexes contain p50 and rel-A
(p65). Nuclear extracts from Stx1-treated THP-1 cells were incubated
with or without rabbit antihuman p50 or p65 antibodies for 10 minutes at room temperature before assessing NF-B binding activity using radiolabeled oligonucleotides. The positions of supershifted bands are
indicated by arrows. The specificity of binding was determined by cold
oligonucleotide competition (lanes 4 and 8).
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AP-1 nuclear translocation by Stx1 in THP-1 cells.
To determine whether AP-1 complex translocation is associated with Stx1
treatment of human monocytes, we examined AP-1 nuclear translocation by
electrophoretic mobility shift assay using radiolabeled target DNA
containing Jun/Jun and Fos/Jun binding motifs. Treatment of cells with
400 ng/mL of Stx1 for 2 hours resulted in translocation of AP-1 into
nuclei (Fig 7). The mutant oligonucleotide
binding (lane 6) and cold oligonucleotide competition (lane 7)
experiments showed that the DNA-protein interaction is AP-1 specific.
TPA and LPS treatments of THP-1 cells were used as the positive
controls of AP-1 translocation.
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| Fig 7.
Stx1-induced nuclear translocation of AP-1 complexes.
Differentiated THP-1 cells were incubated with Stx1 (400 ng/mL), LPS (200 ng/mL), or TPA (200 ng/mL) for 2 hours. Nuclear extracts were
prepared and AP-1 translocation was assessed by electrophoretic mobility shift assay using a radiolabeled consensus AP-1 binding site
probe. Specificity of binding was determined by competition with cold
probe and by the use of an oligonucleotide containing a substitution in
the AP-1 binding motif.
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DISCUSSION |
Given the central role of the host response in the development of
Stx-mediated disease, it would be useful to better understand the
mechanisms by which Stxs trigger cytokine induction to devise interventional strategies limiting the development of life-threatening sequelae. We show here that the incubation of the THP-1 cell line with
purified Stx1 resulted in a dose-dependent increase in TNF bioactivity
detectable in cell supernatants. Northern blot analyses showed that
increased soluble TNF activity was mediated, at least in part, by
transcriptional activation of the gene encoding TNF-. For
comparative purposes, we also treated THP-1 cells with LPS, bacterial
outer membrane constituents that have been shown to possess potent
TNF-inducing capabilities. LPS or Stx 1 treatment stimulated TNF
production by THP-1 cells with dissimilar dose responses; approximately
fourfold more Stx1 was necessary to induce approximately 75% of the
TNF bioactivity induced LPS. In accordance with earlier studies, we
found that LPS induced a rapid transient increase in TNF-
transcripts that returned to basal levels within 3 hours of LPS
stimulation. In contrast, TNF- transcripts produced by Stx1
stimulation reached peak levels more slowly and remained elevated for
longer periods of time (Fig 3).
When THP-1 cells were incubated with Stx1 and LPS, we noted a marked
enhancement in TNF- gene transcription and protein production. Although the precise mechanism of this augmented response remains to be
defined, earlier studies demonstrated that treatment of macrophages
with LPS and the protein synthesis inhibitor, cycloheximide, markedly
upregulated TNF- production, a phenomenon referred to as
superinduction.25 Cycloheximide may inhibit the de novo
synthesis of LPS-induced derepressor molecules or endogenous
endonucleases involved in the degradation of cytokine
transcripts.26-28 We show here that treatment of monocytic
cells with LPS + Stx1 also results in a TNF superinduction effect.
Reagents that simply bind Gb3 (purified Stx1 B-subunits or
monoclonal anti-Gb3 antibody) do not trigger TNF synthesis.
Whether the Stx1 A-subunit and its associated protein synthesis
inhibitory activity are necessary for TNF induction and the LPS + Stx1
TNF superinduction effect is currently under investigation. Anti-LPS
antibodies are frequently detected in HUS patients,29 a
finding consistent with E coli- or Shigella
dysenteriae-mediated gut mucosal damage. Thus, the presence of Stx
and LPS in the circulation may stimulate proinflammatory cytokine
production in a synergistic manner.
In unstimulated macrophages, the five members of the rel family of
transcriptional activators form homodimers or heterodimers that are
retained in the cytoplasm in an inactive state through association with
one of a series of proteins called IB. Upon stimulation with LPS,
proinflammatory cytokines, or phorbol diesters, IB proteins are
phosphorylated, dissociate from NF-B complexes, and undergo
degradation. IB dissociation shows nuclear translocation motifs on
the dimers that then bind with high affinity to DNA containing NF-B
consensus sequences GGGRNNYYCC (where R = purines, Y = pyrimidines, and
N = any nucleotide). NF-B binding regulates the transcription of a
large number of murine genes, including genes encoding cytokines,
chemokines, proto-oncogenes, and leukocyte or endothelial cell adhesion
molecules.13 In contrast to the murine TNF- gene, the
role of NF-B in the activation of the human TNF- gene is
controversial. Multiple NF-B binding sites have been characterized
upstream of the human TNF- transcription start site
(TSS30). Goldfield et al31 reported that each
NF-B site could be deleted without affecting LPS induction of human
TNF- gene expression in transiently transfected murine fibroblasts
or monocytes. In contrast, several investigators demonstrated that LPS
treatment of human monocytic cell lines resulted in the nuclear
translocation of p65/p50 heterodimers and transcriptional
activation.24,32-34 In this study we show that purified
Stx1, like LPS, rapidly (within 60 minutes) stimulates the nuclear
translocation of p65/p50 NF-B complexes to PBMn and THP-1 nuclei and
the degradation of cytoplasmic IB-. Examination of the kinetics
of Stx1 induced NF-B activation shows that nuclear translocation
precedes TNF protein and transcript synthesis. We did not detect the
translocation of p50 homodimers or p50/c-rel complexes in
Stx1-stimulated THP-1 cells.
AP-1 complexes are sequence-specific transcriptional activators
composed of homodimers or heterodimers of the Fos and Jun family of
leucine zipper-containing proteins. The human TNF- gene contains an
AP-1 binding site 59 bp upstream of the TSS, and a number of studies
suggest that AP-1, in combination with other transcriptional
activators, is required for optimal gene expression. For example, the
human TNF- promoter contains a cAMP responsive element (CRE) 100 bp
upstream of the TSS, and treatment of THP-1 cells with LPS resulted in
the induction of TNF- secretion and the selective binding of Jun/ATF
heterodimers at the AP-1/CRE site.35 Recently, Yao et
al36 demonstrated that multiple activators binding to
AP-1/CRE, NF-B (B-3), and Sp1/Egr-1 sites are necessary for
maximal LPS induction of TNF- gene expression in THP-1 cells. Mackman et al37 showed that maximal activation by LPS of
the gene encoding human tissue factor in THP-1 cells required the nuclear translocation of both NF-B and AP-1. Thus, the
transcriptional activation of eukaryotic genes may require cooperative
binding of multiple activators at multiple cis-active sites
within promoter regions.38 We show here that treatment of
THP-1 cells with Stx1 activates the nuclear translocation of factors
capable of binding NF-B and AP-1 consensus sequences.
Although our studies are the first to show that Stxs may induce TNF
production via a transcriptional activation mechanism involving NF-B
and AP-1, a number of infectious agents or toxins have been reported to
trigger nuclear translocation of transcriptional activators. Invasion
of HeLa cell monolayers by Shigella flexneri enhanced protein
binding to AP-1, CREB, and NF-B specific probes.39 Spirochetal lipoproteins will trigger NF-B translocation in THP-1 cells.40 Trede et al41,42 demonstrated that
treatment of THP-1 cells with staphylococcal enterotoxin A (SEA)
induced nuclear translocation of both NF-B (p65/p50) and AP-1
complexes. Interestingly, p65/p50 heterodimers activated by SEA
treatment appeared to preferentially bind the NF-B binding site most
proximal to the TNF- TSS, whereas LPS treatment of Mono Mac 6 cells
resulted in p65/p50 complexes binding to the NF-B site most distal
to the TSS.32 Staphylococcal superantigens are thought to
signal cells via interaction with MHC class II molecules, whereas LPS
has been shown to signal monocytes through CD14 or other membrane
proteins. Whether Stx1, which binds to a membrane glycolipid, also uses
intracellular signaling mechanisms activated by LPS or other bacterial
products and whether Stx1 selectively triggers translocation of p65/p50
complexes to bind to specific NF-B binding sites are currently being
studied.
| |
FOOTNOTES |
Submitted September 8, 1997;
accepted March 12, 1998.
Supported by US Public Health Service Grant No. AI34530.
Address reprint requests to Vernon L. Tesh, PhD, Department of Medical
Microbiology and Immunology, Texas A&M University Health Science
Center, College Station, TX 77843-1114.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank David Acheson and Nancy Rice for sharing reagents
necessary to perform these studies, Bharat Aggarwal for assistance with
the electrophoretic mobility shift assays, and Gregory Foster for
excellent technical assistance.
| |
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Abstract
Introduction
Abstract