| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Gene Expression and Activation of the Transcription Factors NF-
B and Activator Protein-1 in Human Cord Blood Monocytes1
,
,§,,§,,§
*
Infectious Diseases Section, Department of Pediatrics; Cardiology Section, Department of Medicine; and
Winters Center for Heart Failure Research,
Veterans Affairs Medical Center,
§
Baylor College of Medicine, Houston, TX 77030
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is central to the
pathogenesis of septic shock induced by group B
Streptococcus (GBS). In vitro studies using human cord
blood monocytes have demonstrated that GBS induces TNF- secretion,
but little is known about the intracellular signaling pathways of
TNF- induction. In this report we show that heat-killed serotype III
GBS induces host cell signal transduction pathways that lead to
activation of the transcription factors NF-
B and AP-1. Using
adenoviral transfer of IB (IB overexpression), the
production of TNF- induced by whole GBS was inhibited by only 20%.
We also show that the p38 mitogen-activated protein kinase (MAPK)
pathway is involved in GBS-induced TNF- secretion, because TNF-
protein and mRNA levels in the presence of a specific inhibitor of p38
MAPK, SB 202190, were dramatically diminished. EMSAs showed that SB
202190 inhibited GBS-induced AP-1 activation, but had no effect on
NF-B-DNA binding activity. These results indicate that both NF-B
and AP-1 (via p38 MAPK) are involved in the regulation of TNF-
production in GBS-stimulated neonatal monocytes. Therefore, disrupting
the signal transduction pathways induced by GBS has the potential to
attenuate the production of immune response mediators, thereby halting
or possibly reversing the course of this potentially fatal
disease. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Supporting this
model, passive immunization against TNF- affords significant
protection from an otherwise lethal infection with GBS (4, 5). Based on these findings, several investigators have proposed
that optimal therapy of human neonatal GBS sepsis may require
administration of antibiotics and Abs directed against TNF-
(4, 5). However, similar experimental results with
endotoxin-induced shock have not translated into beneficial outcomes in
human clinical trials of anticytokine therapy in Gram-negative sepsis
(6, 7, 8). Therefore, in an effort to develop more effective
strategies to treat patients with sepsis, investigators have sought to
understand the molecular mechanisms by which specific bacteria induce
TNF- production.
To date, the majority of studies have focused on the role of
LPS-induced TNF- synthesis in the setting of Gram-negative sepsis
(9, 10). These studies have shown that LPS is sufficient
to induce TNF- biosynthesis through a pathway that involves p38
mitogen-activated protein kinase (MAPK) and increased activation of
NF-B and AP-1 (11, 12, 13). However, little is known about
the basic mechanisms that are responsible for TNF- biosynthesis
following human infection with Gram-positive organisms such as GBS.
Therefore, in the present study we sought to determine the molecular
mechanisms responsible for GBS-induced TNF- production following
stimulation of human umbilical cord blood monocytes. We show that GBS
interaction with cord blood monocytes induces host cell signal
transduction pathways that result in activation of the transcription
factors NF-B and AP-1 through a pathway that involves
phosphorylation of the p38 MAPK. Thus, these studies suggest that
Gram-positive and Gram-negative bacteria may activate similar signal
transduction pathways in the setting of human sepsis. Accordingly,
these studies raise the interesting possibility that it may be feasible
to develop new therapeutic strategies that may be effective as
adjunctive therapy in both Gram-positive and Gram-negative sepsis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
An encapsulated type III GBS strain (COH1) originally isolated
from the blood of an infant with GBS sepsis was used in the experiments
(provided by Dr. Craig J. Rubens, University of Washington, Seattle,
WA). Bacteria were grown to log phase in Columbia broth and heat killed
by incubating the organisms at 56°C for 1 h. Aliquots were
stored at -70°C for later use. At a bacterial concentration of
108 CFU/ml, the endotoxin contamination was 
10
pg/ml as measured by the Limulus amebocyte lysate assay
(Chromogenic LAL, BioWhittaker, Walkersville, MD; test performed by Dr
Edward O. Mason, Jr., C. T. Parker Laboratory, Texas Children’s
Hospital, Houston, TX).
Isolation of human umbilical cord blood monocytes
Umbilical cord blood was collected immediately after delivery of
the placenta during uncomplicated elective cesarean section. Monocytes
were purified by gradient density centrifugation and enriched by
adherence to tissue culture plates. As previously reported by our group
(14), this technique results in an adherent cell
population consisting of 85–90% monocytes. Viability, determined by
trypan blue exclusion, was consistently >95%. Cells were maintained
in RPMI 1640 (Life Technologies, Gaithersburg, MD) tissue culture
medium supplemented with L-glutamine, 1% heat-inactivated
human serum, 25 mM HEPES, and gentamicin (50 µg/ml). All reagents and
culture media used in monocyte isolation and stimulation studies
contained <0.03 endotoxin units/ml by Limulus amebocyte
lysate assay (Associates of Cape Cod, Woods Hole, MA). Monocytes were
cultured at a density of 107 cells/ml for mRNA
extraction, nuclear extraction experiments, adenoviral infection
studies, p38 MAPK measurements, and IB- measurements. A total of
106 cells/ml were stimulated for TNF- protein
production. For all experiments monocytes were incubated at 37°C in a
5%CO2 atmosphere.
Adenoviral vectors and their propagation
An adenovirus encoding porcine IB (AdvIB) with a CMV
promoter and a nuclear localization sequence was provided by Dr. R. de
Martin (Vienna, Austria). A recombinant, replication-deficient,
adenoviral vector encoding Escherichia coli
ß-galactosidase (Adv-ßgal) was provided by Dr. Alan Davis (Center
for Cell and Gene Therapy, Baylor College of Medicine). Vector
propagation and titrating were performed in the 293 human embryonic
kidney cell line at the Baylor Center for Cell and Gene Therapy using
standard methods (15).
Infection techniques
Infection of monocytes was performed exactly as described by
Bondeson et al. (16). The isolated monocytes were
incubated at 107/ml in RPMI 1640 with 25 mM HEPES
and 2 mM L-glutamine supplemented with 5% (v/v)
heat-inactivated FCS and 50 µg/ml gentamicin. The isolated monocytes
were pretreated with macrophage CSF (100 ng/ml; R&D Systems,
Minneapolis, MN) for 48 h. The cells were subsequently infected
for 2 h with a multiplicity of infection of 80:1 (determined in
dose-response experiments) of either AdvIB or Adv-ßgal in
serum-free RPMI 1640. Cells were then incubated in RPMI supplement as
described above for 48 h to allow significant overexpression of
IB. The efficiency of infection was assessed by expression of
ß-gal using the ß-gal staining kit as recommended by the
manufacturer (Invitrogen, Carlsbad, CA).
Stimulation of cells
Monocytes were stimulated for various time periods with
heat-killed, but intact, serotype III GBS (108
CFU/ml) in the presence of 5% heat-inactivated human serum.
Supernatants for TNF- protein determination were harvested at 0, 30,
60, 120, and 240 min. Total RNA was isolated at 4 h, and nuclear
protein extracts were prepared at 0, 30, and 60 min after GBS
stimulation of monocytes. For NF-B studies, monocytes stimulated for
60 min with LPS E. coli strain 055:B5 (1 µg/ml; Sigma, St.
Louis, MO) or recombinant human TNF- (25 ng/ml; R&D Systems) were
used as controls. For IB- degradation and p38 MAP kinase
activity, cells were stimulated from 0–30 min. To evaluate the role of
the p38 MAPK pathway in GBS-induced TNF- production, monocytes were
pretreated (1 h) with varying concentrations of SB 202190 (Calbiochem,
La Jolla, CA) and subsequently stimulated with GBS for 4 h. SB
202190 is a cell-permeable pyridinyl imidazole that acts as an
inhibitor of p38 MAPK without significant effect on the activity of
extracellular regulated kinase or c-Jun amino-terminal kinase
subgroups. This compound has been reported to block LPS-induced TNF-
and IL-1ß production in mice (17).
TNF- ELISA
To detect TNF- production, monocytes
(106 cell/ml) were suspended in RPMI 1640
containing 5% heat-inactivated human serum. Cells were subsequently
stimulated with GBS (108 CFU/ml) at 37°C for
various times. Cell-free supernatants were harvested and analyzed for
TNF- production using a commercially available ELISA kit (Genzyme,
Cambridge, MA). Samples were quantified by reference to a standard
curve constructed using human recombinant TNF- standards (15–1200
pg/ml). Results were expressed as the mean ± SEM of three
independent experiments.
RNA isolation and Northern blot analysis
Total RNA was extracted from monocytes
(107 cell/ml) by the guanidinium thiocyanate
method (18). Total RNA (5 µg/lane) was denatured at
90°C for 5 min, size fractionated on a 1% agarose gel containing 2.2
M formaldehyde, transferred onto a nylon membrane (Gene-Screen,
DuPont-NEN, Boston, MA), and hybridized sequentially to random primed
cDNA probes. The following probes were used for Northern blot analyses:
a 1.1-kb PstI fragment of human TNF- (American Tissue
Culture Collection, Manassas, VA) and a 0.5-kb
Xba/HindIII fragment of human GAPDH gene, which
was used as an internal control. Autoradiograms were prepared by
exposure of blots to Hyperfilm MP (Amersham Pharmacia Biotech,
Piscataway, NJ) at -70°C. The signals were quantified with
ImageQuant software (Personal Densitometer I, Molecular Dynamics,
Sunnyvale, CA).
Preparation of nuclear protein extracts
At the indicated intervals after stimulation, nuclear extracts were prepared as described by Tran-Thi et al. (19). In brief, cord blood monocytes were washed with cold PBS and suspended in 1 ml of buffer. After 15 s of centrifugation at 14,000 x g, cell pellets were lysed with 35 µl of cold buffer C (20 mM HEPES-NaOH (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT). After 30 min on ice, cell lysates were centrifuged at 14, 000 x g at 4°C for 15 min. The nuclear proteins were quantified by the bicinchoninic acid method (Pierce, Rockford, IL) and stored in aliquots at -70°C.
Electrophoretic mobility shift assays
The NF-B consensus oligonucleotide probe
(5'-AGTTGAGGGGACTTTCCCAGGC-3') and the AP-1 oligonucleotide probe
(5'-CGCTTGATGACTCAGCCGGA-3') were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). The probes were labeled with
[
-32P]ATP using T4 polynucleotide kinase
(Life Technologies) and were purified in Bio-Spin chromatography
columns (Bio-Rad, Hercules, CA). For EMSA, 10 µg of nuclear proteins
were preincubated with EMSA buffer (12 mM HEPES-NaOH (pH 7.9), 60 mM
KCl, 1 mM EDTA, 1 mM DTT, 2 µg poly(dI-dC), and l0% glycerol) on ice
for 10 min before addition of the radiolabeled probe for an additional
30 min at room temperature. Competition studies were conducted with 50-
and 100-fold molar excesses of unlabeled oligonucleotides added to the
reaction mixtures before addition of the radiolabeled oligonucleotides.
DNA-protein complexes were resolved by electrophoresis on native 4%
polyacrylamide gels in 0.5x Tris borate-EDTA buffer for 2 h at
160 V. Gels were transferred to Whatman 3M paper (Whatman, Clifton,
NJ), dried under a vacuum at 80°C for 2 h, and exposed overnight
to Hyperfilm MP (Amersham Pharmacia Biotech) at -70°C with an
intensifying screen.
IB Western blot analysis
Cytoplasmic extracts were prepared from
107 cell/ml at different times after stimulation
with GBS. Cells were lysed with lysis buffer (50 mM Tris-HCl; 150 mM
NaCl; 1% Triton X-100 (pH 8.0); 1 µg/ml each of leupeptin,
pepstatin, and antipain; 1 mM PMSF; 1 mM NaF; and 1 mM
Na3VO4), and protein
concentrations were determined using the bicinchoninic acid method
(Pierce). Whole cell lysates were boiled in equal volumes of loading
buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and 10%
2-ME), and 30 µg of protein was loaded per lane in 12%
polyacrylamide gels. Proteins were separated electrophoretically and
transferred to nitrocellulose membranes (Bio-Rad) using the Bio-Rad
MiniGel system. For immunoblotting, membranes were blocked with 5%
nonfat dried milk in Tris-buffered saline (25 mM Tris buffer (pH 7.6)
containing 137 mM NaCl) with 0.05% Tween 20 (TBST) for 1 h.
Immunostaining for IB- was performed with polyclonal rabbit
anti-human IB- (diluted 1/1000; Santa Cruz Biotechnology) Ab.
After washing three times with TBST, the blots were incubated with a
1/1000 dilution of secondary Ab consisting of HRP-conjugated
anti-rabbit IgG (Amersham Pharmacia Biotech) Ab for 2 h. Blots
were washed three times with TBST, incubated in enhanced
chemiluminescence reagents (ECL-Plus, Amersham Pharmacia Biotech), and
exposed to photographic film.
p38 MAPK Western blot analysis
Protein phosphorylation of p38 has been shown to be an accurate indicator of its activation (20). To determine the protein phosphorylation of p38 MAPK at different times after stimulation with GBS, monocytes (107 cells/ml) were washed with cold PBS containing 1 mM Na3VO4 and subsequently lysed in lysis buffer (50 mM Tris-HCl; 150 mM NaCl; 1% Triton X-100 (pH 8.0); 1 µg/ml each of leupeptin, pepstatin, and antipain; 1 mM PMSF; 1 mM NaF; and 1 mM Na3VO4). Proteins were separated by 12% SDS-PAGE, transferred, and blocked as described above. Membranes were subsequently incubated with 1/500 diluted primary rabbit Ab against Thr180/Tyr182-phosphorylated p38 MAPK (New England Biolabs, Beverly, MA) at 4°C overnight. After washing three times with TBST, the blots were incubated with 1/1000 diluted secondary Ab of HRP-conjugated anti-rabbit IgG (Amersham Pharmacia Biotech) for 2 h. Blots were washed three times with TBST, incubated in enhanced chemiluminescence reagents (ECL-Plus, Amersham Pharmacia Biotech), and exposed to photographic film. Membranes were also probed with primary Ab raised against unphosphorylated p38 MAPK (New England Biolabs).
Statistical analysis
The results represent the mean ± SEM of three experiments
unless otherwise specified. One-way ANOVA was used to test for mean
differences in TNF- production after treatment with the p38 MAPK
inhibitor SB 202190. A p value <0.05 was considered
significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
after stimulation of cord blood monocytes with
GBS
The kinetics of TNF- production were examined
(n = 3) by incubating monocytes with GBS
(108 CFU/ml) for varying time points (Fig. 1
A). Measurable TNF-
protein biosynthesis in the supernatant was detected within 2 h of
exposure and increased dramatically by 4 h. Unstimulated monocytes
(0, 0.5, 1, 2, and 4 h) and those stimulated for 30–60 min
produced low, but detectable, amounts of TNF-. This finding is in
accordance with previous studies reported from this laboratory
(14).
|
gene expression with the appearance
of TNF- in the supernatant of stimulated cells (Fig. 1
B).
An increase (from baseline) in TNF- mRNA expression was first
detected 1 h after stimulation with GBS and increased dramatically
by 4 h. These data suggested that GBS-induced TNF- biosynthesis
was at least in part regulated at the transcriptional level.
Activation of transcription factors NF-B and AP-1 by GBS
Incubation of GBS with cord blood monocytes may induce alterations
in signal transduction pathways that modulate cellular transcription
factors. Furthermore, the activation of transcription factors and
proinflammatory cytokines such as TNF- may be coordinately inducible
in GBS-stimulated monocytes. To elucidate the mechanism(s) by which GBS
induces the transcriptional activity of the TNF- gene, we analyzed
the binding activities of NF-B and AP-1 in GBS-exposed monocytes by
EMSA. Cells treated with TNF- or LPS served as positive controls. At
different time points after challenge, nuclear extracts were assayed
for NF-B-DNA binding activity using a radiolabeled specific
oligonucleotide probe. As shown in Fig. 2A (n = 3),
nuclear extracts from unstimulated monocytes contained low basal levels
of NF-B binding activity. In accordance with the kinetics of
GBS-induced TNF- biosynthesis and TNF- mRNA induction, inducible
NF-B binding activity was detected within 30 min and increased
progressively (60 min) following exposure to GBS. Cold oligonucleotide
competition demonstrated that the binding of NF-B was specific, as
the unlabeled NF-B oligonucleotide prevented formation of
radiolabeled protein-DNA complexes. As another measure of NF-B
activation we examined the changes in cytoplasmic IB (37-kDa)
protein levels in GBS-stimulated monocytes. Immunoblotting analysis
revealed that GBS rapidly induced the disappearance of IB within
15–30 min (Fig. 2B) after stimulation of monocytes.
|
B represents a critical event in the induction TNF- by
heat-killed GBS. To further define the role of NF-B in the
downstream signaling after exposure to GBS, we asked whether IB
overexpression (AdvIB) could block TNF- mRNA up-regulation and
protein biosynthesis. As shown in Fig. 3A, overexpression of IB
abrogated (100%; n = 3) NF-B-DNA binding in
monocytes stimulated with either GBS or LPS. However, IB
overexpression decreased GBS-induced TNF- mRNA up-regulation and
protein biosynthesis by only 15 and 20%, respectively (Fig. 3, B and C). In contrast, IB overexpression
inhibited LPS-induced TNF- mRNA and protein up-regulation by 70 and
65%, respectively (Fig. 3, B and C).
Interestingly, in parallel experiments the proteosome inhibitors MG-132
and N-tosyl-L-phenylalanine
chloromethyl ketone abrogated GBS-induced NF-B activation and
decreased both TNF- gene expression and protein synthesis by >85%
(data not shown).
|
). Cold oligonucleotide competition
experiments showed that the DNA-protein interaction was AP-1
specific.
|
Various members of the MAPK family of proteins may modulate
regulation of AP-1 activity through phosphorylation (21).
In addition, recent studies have suggested that the p38 MAPK pathway
may be involved in the activation of NF-B (22, 23).
Therefore, we examined the capacity of GBS to induce activation of the
p38 MAPK pathway in stimulated monocytes. As shown in Fig. 5A, cellular p38 MAPK became
phosphorylated (and hence activated) in a time-dependent manner in
monocytes following stimulation with GBS. The findings were confirmed
by examining total cellular p38 MAPK, and Fig. 5A indicates
that the differences observed for the induced protein phosphorylation
of the cellular MAPK did not result from differences in loading or from
cellular protein digestion.
|
induction, a specific inhibitor, SB 202190, was
used to block p38 MAPK activity. At concentrations ranging from
0.1–100 µM, this compound has been shown to effectively inhibit
phosphorylation of myelin basic protein and ATF2 by recombinant p38
(24). As shown in Fig. 5B, stimulation of
monocytes with GBS in the absence of SB 202190 led to a significant
increase in TNF- production. Addition of SB 202190 reduced TNF-
secretion following stimulation with GBS by 90%
(p 0.05, 5 µM SB 202190 vs no inhibitor).
To further investigate the molecular mechanisms of the regulation of
TNF- expression by the p38 MAPK pathway, TNF- mRNA levels were
studied by Northern blot analyses in GBS-exposed monocytes in the
absence or the presence of SB 202190 (5 µM). Fig. 5C shows
that treatment of cells with 5 µM SB 202190 resulted in an 86%
reduction (normalized for GAPDH) in TNF- transcripts induced by GBS.
Taken together, these data demonstrate that the p38 MAPK pathway plays
a key role in mediating GBS-induced TNF- production by cord blood
monocytes.
Effect of SB 202190 on GBS-induced AP-1 and NF-B activation
The above data demonstrate a transcriptional control of TNF- by
p38 MAPK in GBS-stimulated monocytes. Recent studies have suggested a
possible role for p38 MAPK in the regulation of AP-1 and NF-B
(23). We therefore investigated the effect of SB 202190 on
GBS-induced AP-1and NF-B activation monocytes. Cord blood monocytes
were treated with SB 202190 (5 µM) and subsequently stimulated with
GBS for 60 min. As shown in Fig. 6A, SB 202190 completely
inhibited the GBS-induced activation of AP-1, as determined by EMSA. In
an identical nuclear extract used in AP-1 studies, it was found that SB
202190 was not able to prevent GBS-induced NF-B-DNA binding activity
(Fig. 6B), suggesting that inhibition of the p38 MAPK
pathway did not interfere with release of NF-B from IB.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in the development and
progression of GBS septic shock, it is important to understand the
mechanisms by which this bacterium triggers TNF- production. We show
here that GBS stimulation of cord blood monocytes results in a
dose-dependent increase in immunoreactive TNF- detectable in cell
supernatants. In accordance with earlier studies from this laboratory
(14), substantial TNF- secretion by monocytes was only
detected after 2 h of incubation (Fig. 1A). Northern
blot analyses suggested that the GBS-induced increase in TNF-
production was mediated at least in part by transcriptional activation
of the gene encoding TNF- (Fig. 1B). Furthermore, the
observed increase in TNF- transcripts in GBS-stimulated monocytes
was accompanied by the activation of the transcription factors NF-B
(Fig. 2A) and AP-1 (Fig. 4). Analysis of the cytoplasmic
levels of the NF-B inhibitory protein IB showed that
stimulation with GBS caused its disappearance within 15–30 min (Fig. 2B). To substantiate a possible relation between NF-B
activation and GBS-induced TNF- production, IB was
overexpressed (Adv-IB) in monocytes before stimulation with GBS.
Under these conditions, NF-B-DNA binding activity was markedly
reduced following exposure to GBS. However, TNF- mRNA expression and
protein synthesis were only minimally affected. In contrast, as
previously reported (16), IB overexpression
inhibited LPS-induced NF-B activation and significantly
reduced TNF- biosynthesis. These findings suggest that
transcription factors other than NF-B may also play an
important role in the induction of TNF- by whole GBS in human
monocytes.
Indeed, our studies also show that GBS induces activation of the
transcription factor AP-1 through a p38-dependent pathway. AP-1
complexes are sequence-specific transcriptional activators composed of
homodimers or heterodimers of the Fos and Jun families of leucine
zipper-containing proteins (20). The human TNF- gene
contains an AP-1 binding site, and a number of studies suggest that
AP-1, in combination with other transcriptional activators, is required
for optimal gene expression. Recently, studies by Yao et al.
(25) have demonstrated that multiple activators binding to
AP-1/cAMP response element, NF-B, and Sp1/Erg-1 sites are necessary
for maximal LPS induction of TNF- gene expression in the human
monocytic cell line THP-1. Mackman et al. (26) also
reported that maximal LPS-induced expression of the human tissue factor
gene in THP-1 cells required activation of both NF-B and AP-1. In
addition, Wang et al. (27) recently reported that
peptidoglycan from Staphylococcus aureus induces activation
of the transcription factors cAMP response element binding protein/ATF
and AP-1 in THP-1 cells via a CD14-dependent pathway.
Medvedev et al. (28) have recently demonstrated that
GBS cell wall extracts induce NF-B activation and TNF- production
in CHO cells expressing CD14; however, the upstream components involved
in GBS-induced-NF-B or AP-1 activation have not been elucidated. In
this study we also analyzed the capacity of heat-killed, but intact,
GBS to induce stress response kinase pathways leading to NF-B and
AP-1 activation in cord blood monocytes. There are at least three main
groups of mitogen-activated protein kinases: extracellular
signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. In
general, the ERKs are largely involved in pathways leading to cell
proliferation as a consequence of growth factor stimulation, whereas
JNK and p38 MAPK are activated in response to a variety of cytokines
and stress conditions (29). In particular, we studied the
p38 MAPK pathway, as recent studies have suggested its involvement in
the regulation of both NF-B and AP-1 (23). We found
that GBS induces rapid phosphorylation of p38 in cord blood monocytes
(Fig. 5A). The effect of SB 202190 demonstrated that
activation of this signaling pathway is necessary for TNF- induction
by GBS. This compound decreased TNF- mRNA accumulation by 86% (Fig. 5C) and TNF- biosynthesis by 90% in stimulated cells. In
agreement with previous reports indicating that p38 MAPK is required
for the activation of several transcription factors, including cAMP
response element-binding protein (30), c-Fos, and c-Jun
(21), we have shown that GBS induction of AP-1 requires
activation of the p38 MAPK pathway. Thus, the effect of SB 202190 on
TNF- mRNA (Fig. 5C) is probably due in part to a decrease
in the abundance of the AP-1 components c-Fos and c-Jun. Furthermore,
recent studies have suggested that p38 MAPK plays an important role in
mRNA stabilization in primary monocytes stimulated with LPS
(31). It is possible that instability caused by SB 202190
could have contributed to the observed decrease in mRNA after GBS
stimulation. In contrast, inhibition of p38 MAPK had no effect on
NF-B-DNA binding activity as detected by EMSA (Fig. 6B).
Very recently, several investigators have shown that SB 203580 (a p38
inhibitor) or a dominant-negative mutant of MAPK kinase-6 is capable of
blocking NF-B-mediated luciferase trans-activation in
response to TNF- without affecting NF-B translocation (23, 32). Furthermore, Garcia et al. (33) also have
shown that the p38 MAPK pathway is required for NF-B activation in
macrophages stimulated with lipid-associated membrane proteins from
Mycoplasma fermentans. Together, our results and those of
others underscore the important role that p38 MAPK plays in cytokine
regulation and nuclear responses following stimulation with
Gram-positive and Gram-negative bacteria (34, 35).
In conclusion, our data agree with and extend earlier findings
concerning the molecular mechanisms by which GBS induces the production
of TNF- (28). It remains to be determined whether CD14
is required for NF-B/AP-1 activation and TNF- production in human
neonatal monocytes stimulated with whole GBS. Preliminary data from our
laboratory suggest that whole GBS and its cell wall components induce
TNF- production in human neonatal monocytes via a CD14-independent
pathway. In addition, Cohen et al. (36) recently reported
that mice deficient in CD14 expression are not resistant to GBS
infection and actually have higher serum TNF- levels than wild-type
animals. Thus, elucidating the signaling pathways activated by specific
pathogens is the most accurate means for analyzing the steps involved
in the induction of the inflammatory response. As shown here,
inhibition of the p38 MAPK pathway by the synthetic compound SB 202190
effectively blocked monocyte signal transduction pathways involved in
the initiation of the acute inflammatory response induced by
heat-killed GBS. Therefore, SB 202190 and other inhibitors of signal
transduction (i.e., protein tyrosine kinase inhibitors) have the
therapeutic potential to interfere with the cascade of events leading
to septic shock and end-organ dysfunction. The use of such agents could
be effective for a variety of both Gram-positive and Gram-negative
bacteria, especially in the early stages of infection, when the
identity of the etiologic agent is not known.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Jesus G. Vallejo, Department of Pediatrics, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030.
3 Abbreviations used in this paper: GBS, group B Streptococcus; MAPK, mitogen-activated protein kinase; AdvIB, adenovirus encoding porcine IB; Advßgal, adenovirus encoding Escherichia coli ß-galactosidase; ß-gal, ß-galactosidase.
Received for publication March 1, 1999. Accepted for publication April 13, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and interleukin-6 in mice infected with group B streptococci. Circ. Shock 38:138.[Medline]
in a neonatal rat model of group B streptococcal infection. Infect. Immun. 61:227.: use as adjunctive therapy in established group B streptococcal disease in newborn rats. Pediatr. Res. 38:551.[Medline]
in patients with sepsis syndrome. JAMA 273:934. transcription in macrophages: involvement of four B like motifs and of constitutive and inducible forms of NF-B. Infect. Immun. 10:1498.
by type III group b streptococci. Infect. Immun. 64:5042.[Abstract]
B into human macrophages: lipopolysaccharide-induced, but not zymosan-induced, proinflammatory cytokines are inhibited, but IL-10 is nuclear factor-B independent. J. Immunol. 162:2939.B and AP-1 in rat liver macrophages. Hepatology 22:613.[Medline]
B and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways. J. Biol. Chem. 272:12422.
. J. Biol. Chem. 272:30122. promoter in human monocytic cells: regulation by Erg-1, c-Jun, and NF-B transcription factors. J. Biol. Chem. 272:17797.
B binding sites. J. Exp. Med. 174:1517.B activation and TNF production induced by lipopolysaccharide and group b streptococcal cell walls. J. Immunol. 160:4535.B degradation and nuclear factor-B DNA binding are insufficient for interleukin-1ß and tumor necrosis factor- induced B-dependent transcription. J. Biol. Chem. 273:6607.B activity and cytokine secretion in macrophages via activation of mitogen-activated protein kinase pathways. J. Biol. Chem. 273:34391. accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-. J. Infect. Dis. 179:939.
response, but plays no major role in shock induced by group B Streptococcus infection. Pediatr. Res. 45:267A. (Abstr. 1574).
This article has been cited by other articles:
|
|
 |
|
  S. Kenzel, S. Santos-Sierra, S. D. Deshmukh, I. Moeller, B. Ergin, K. A. Fitzgerald, E. Lien, S. Akira, D. T. Golenbock, and P. Henneke Role of p38 and Early Growth Response Factor 1 in the Macrophage Response to Group B Streptococcus Infect. Immun., June 1, 2009; 77(6): 2474 - 2481. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kenzel, G. Mancuso, R. Malley, G. Teti, D. T. Golenbock, and P. Henneke c-Jun Kinase Is a Critical Signaling Molecule in a Neonatal Model of Group B Streptococcal Sepsis. J. Immunol., March 1, 2006; 176(5): 3181 - 3188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Levy, R. M. Jean-Jacques, C. Cywes, R. B. Sisson, K. A. Zarember, P. J. Godowski, J. L. Christianson, H.-K. Guttormsen, M. C. Carroll, A. Nicholson-Weller, et al. Critical Role of the Complement System in Group B Streptococcus-Induced Tumor Necrosis Factor Alpha Release Infect. Immun., November 1, 2003; 71(11): 6344 - 6353. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Lagoumintzis, M. Christofidou, G. Dimitracopoulos, and F. Paliogianni Pseudomonas aeruginosa Slime Glycolipoprotein Is a Potent Stimulant of Tumor Necrosis Factor Alpha Gene Expression and Activation of Transcription Activators Nuclear Factor {kappa}B and Activator Protein 1 in Human Monocytes Infect. Immun., August 1, 2003; 71(8): 4614 - 4622. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ghosh, S. Bhattacharyya, M. Sirkar, G. S. Sa, T. Das, D. Majumdar, S. Roy, and S. Majumdar Leishmania donovani Suppresses Activated Protein 1 and NF-{kappa}B Activation in Host Macrophages via Ceramide Generation: Involvement of Extracellular Signal-Regulated Kinase Infect. Immun., December 1, 2002; 70(12): 6828 - 6838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N.-H. Cho, S.-Y. Seong, M.-S. Huh, N.-H. Kim, M.-s. Choi, and I.-s. Kim Induction of the Gene Encoding Macrophage Chemoattractant Protein 1 by Orientia tsutsugamushi in Human Endothelial Cells Involves Activation of Transcription Factor Activator Protein 1 Infect. Immun., September 1, 2002; 70(9): 4841 - 4850. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Mancuso, A. Midiri, C. Beninati, G. Piraino, A. Valenti, G. Nicocia, D. Teti, J. Cook, and G. Teti Mitogen-Activated Protein Kinases and NF-{kappa}B Are Involved in TNF-{alpha} Responses to Group B Streptococci J. Immunol., August 1, 2002; 169(3): 1401 - 1409. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Knuefermann, P. Chen, A. Misra, S.-P. Shi, M. Abdellatif, and N. Sivasubramanian Myotrophin/V-1, a Protein Up-regulated in the Failing Human Heart and in Postnatal Cerebellum, Converts NFkappa B p50-p65 Heterodimers to p50-p50 and p65-p65 Homodimers J. Biol. Chem., June 21, 2002; 277(26): 23888 - 23897. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. R. James, D. Tang, A. Ingram, H. Ly, K. Thai, L. Cai, and J. W. Scholey Flux Through the Hexosamine Pathway Is a Determinant of Nuclear Factor {kappa}B- Dependent Promoter Activation Diabetes, April 1, 2002; 51(4): 1146 - 1156. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
