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B Are Involved in TNF-
Responses to Group B Streptococci1
* Department of Experimental Pathology and Microbiology, University of Messina, Messina, Italy; and
Department of Pharmacology and Neuroscience, Medical University of South Carolina, Charleston, SC 29425
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is a mediator of lethality in experimental infections by
group B streptococcus (GBS), an important human pathogen. Little is
known of signal transduction pathways involved in GBS-induced TNF-
production. Here we investigate the role of mitogen-activated protein
kinases (MAPKs) and NF-
B in TNF- production by human monocytes
stimulated with GBS or LPS, used as a positive control. Western blot
analysis of cell lysates indicates that extracellular signal-regulated
kinase 1/2 (ERK 1/2), p38, and c-Jun N-terminal kinase MAPKs, as well
as IB, became phosphorylated, and hence activated, in both LPS-
and GBS-stimulated monocytes. The kinetics of these phosphorylation
events, as well as those of TNF- production, were delayed by 30–60
min in GBS-stimulated, relative to LPS-stimulated, monocytes. Selective
inhibitors of ERK 1/2 (PD98059 or U0126), p38 (SB203580), or NF-B
(caffeic acid phenetyl ester (CAPE)) could all significantly reduce
TNF- production, although none of the inhibitors used alone was able
to completely prevent TNF- release. However, this was completely
blocked by combinations of the inhibitors, including PD98059-SB203580,
PD98059-CAPE, or SB203580-CAPE combinations, in both LPS- and
GBS-stimulated monocytes. In conclusion, our data indicate that the
simultaneous activation of multiple pathways, including NF-B, ERK
1/2, and p38 MAPKs, is required to induce maximal TNF- production.
Accordingly, in septic shock caused by either GBS or Gram-negative
bacteria, complete inhibition of TNF- release may require treatment
with drugs or drug combinations capable of inhibiting multiple
activation pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Increased systemic concentrations of proinflammatory cytokines,
including TNF-, have been correlated with septic shock and mortality
(7, 8, 9). The ability of GBS to induce proinflammatory
cytokine production has been documented using human leukocytes
(10, 11, 12, 13, 14, 15) and rodent infection models
(16, 17, 18, 19, 20). In the latter, TNF- plays a central role in
mediating mortality (16, 18). Therefore, elucidation of
the cellular mechanisms involved in GBS-induced cytokine production may
be important to develop effective therapeutic strategies.
Receptors and signal transduction molecules involved in GBS-induced
TNF- production are poorly understood. The type 3 complement
receptor is involved in such responses, although it is unlikely
that this receptor alone can initiate the signal transduction cascade
leading to TNF- production (13, 15). Toll-like
receptors (TLRs) play a central role in the recognition of
microorganisms (21). TLR4 was shown to mediate responses
to the LPS component of Gram-negative bacteria (22, 23, 24),
whereas TLR2 is involved in the recognition of Gram-positive cell wall
components, zymosan, bacterial lipoproteins, and mycobacterial
components (25, 26, 27, 28, 29).
However, because neither TLR2 (30) nor TLR4
(15) play an obligatory role in cellular activation
induced by whole GBS cells, the role of TLRs in the recognition of
these bacteria still remains to be elucidated. A recent report suggests
that some as yet undefined member of the TLR family may be involved in
responses to GBS, because macrophages from mice with a targeted
disruption of the TLR-associated adaptor protein MyD88 are unable to
produce TNF- responses to GBS (31). Phosphorylation of
proteins at tyrosine residues is apparently involved in GBS-induced
cell activation, as suggested by the ability of a tyrosine kinase
inhibitor to decrease mediator production and enhance survival of rats
after GBS-induced shock (32).
Among the most conserved tyrosine-phosphorylated proteins involved in signal transduction are the mitogen-activated protein kinases (MAPKs), which include extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38. A unique feature of MAPKs is that they become activated after phosphorylation of both their tyrosine and threonine amino acids (dual phosphorylation).
MAPKs are arranged in modules composed of three protein kinases that phophorylate and activate each other sequentially (33). MAPK kinase kinase activates MAPK kinase (MAPKK), which in turn activates MAPK. The ERK pathway, which involves both the p42 and p44 isoforms (ERK1 and ERK2, respectively) is activated by the dual-specificity MAPKK MEK1 and MEK2. Likewise, MKK3 and MKK6 (MAPKK homologs) activate p38.
After activation, MAPKs phosphorylate, and hence activate, both
specific transcription factors (34, 35, 36) and components of
the general transcription machinery (37). Interestingly,
MAPKs are involved in cell activation initiated by a wide spectrum of
microbial stimuli, including LPS and other microbial products
(38, 39, 40, 41, 42). The transcription factor NF-B is also known
to play a central role in the expression of proinflammatory cytokine
genes after exposure to LPS and other microbial stimuli (43, 44). Although previous studies have shown that GBS can induce
the activation of NF-B (15, 45), the causal role of the
latter in GBS-induced cytokine production is unclear.
The present study was conducted to elucidate the role of MAPKs and
NF-B activation in TNF- responses of human monocytes to GBS. Our
results indicate that GBS simultaneously activate multiple pathways and
that the coordinated activation of ERK, p38, and NF-B is required
for maximal TNF- secretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemicals were obtained from Sigma Chimica (Milan, Italy),
unless indicated otherwise. SB203580, caffeic acid phenetyl ester
(CAPE), calphostin C, GÖ 6976, and Ro 31-8220 were obtained from
Calbiochem (La Jolla, CA). PD98059 was purchased from New England
Biolabs (Beverly, MA). U0126 was kindly provided by DuPont
Pharmaceuticals (Wilmington, DE). Goat anti-human TNF- IgG and
control normal goat IgG were purchased from R&D Systems (Minneapolis,
MN). IL-1R antagonist (IL-1ra), a kind gift from Amgen (Boulder, CO),
was stored at -80°C. All cell culture media and reagents were
obtained from Life Technologies (San Guliano Milanese, Italy).
Bacteria
COH1, a highly virulent encapsulated type III GBS strain originally isolated from a septic neonate, was kindly provided by C. Rubens (University of Washington, Seattle, WA). Staphylococcus aureus and Listeria monocytogenes were recent clinical isolates. Bacteria were grown to the early stationary phase in a chemically defined medium (46) and were harvested by centrifugation. Killed bacteria were prepared by heat treatment (80°C for 45 min), followed by extensive washing with distilled water and lyophilization. The endotoxin level of all of the lyophilized bacterial preparations was <0.06 EU/mg, as determined by Limulus amebocyte lysate assay (PBI, Milan, Italy).
TNF- production by human monocytes
Mononuclear cells were obtained by Ficoll-Hypaque density
gradient centrifugation (Amersham Pharmacia Biotech, Milan, Italy)
(47) from the peripheral blood of healthy adult donors or
from the cord blood of term neonates. Cells at the interface were
extensively washed, resuspended in RPMI 1640 medium supplemented with
streptomycin (50 µg/ml) and benzylpenicillin (50 IU/ml) to a
concentration of 1.5 x 106/ml, and cultured
in 24-well culture plates for 2 h at 37°C in 5%
CO2. To remove nonadherent cells, the wells were
washed twice with prewarmed culture medium. Adherent cells (>90%
monocytes) were then stimulated for the indicated times with killed
bacteria or Salmonella enteritidis LPS. Culture supernatants
were collected and stored at -70°C until assayed for TNF-. In
MAPK and NF-B blockade experiments, monocytes were pretreated for
1 h with different inhibitors at the indicated concentrations
before stimulation with the bacterial products. In preliminary
experiments, it was found that cell viability, as determined by trypan
blue exclusion, was not affected by any of the treatments used in the
present study.
Western blot analysis of monocyte lysates
PBMCs were separated from blood as described above and dispensed to six-well tissue culture plates at a concentration of 6 x 106 cells/ml. Adherent cells were collected at various times after the addition of GBS or LPS or plain medium as a control. Monolayers were washed twice with ice-cold PBS supplemented with 1 mM sodium orthovanadate, 1 mM PMSF, and 1 mM NaF and were harvested with a plastic scraper. The cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM EDTA, 10 mM NaF, 1 mM PMSF, and 10 µg/ml each of leupeptin, pepstatin, and aprotinin) by incubation on ice for 30 min. Lysates were then centrifuged at 13,000 x g for 15 min at 4°C. Protein concentration in each sample was determined using a standard Bradford protein assay (Bio-Rad Laboratories, Milan, Italy). A protein sample (30 µg) from each reaction mixture was electrophoresed in 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose membranes (Amersham Pharmacia Biotech), and blocked in TBST supplemented with 5% milk for 1 h. The membranes were subsequently incubated with 1/500-diluted primary phosphospecific rabbit IgGs recognizing Thr180/Tyr182-phosphorylated p38 MAPK, Thr202/Tyr204-phosphorylated p44/42 MAPK, or Thr183/Tyr185-phosphorylated JNK MAPK (New England Biolabs).
Because phosphorylation of IB at Ser32 is
essential for release of active NF-B, phosphorylation at this site
was used as a marker of NF-B activity. To detect phosphorylated
IB, the nitrocellulose membranes were reacted with
phospho-IB-specific rabbit polyclonal IgG (New England Biolabs)
at a 1/500 dilution. After a 4°C overnight incubation, membranes were
washed with TBST and incubated with donkey anti-rabbit IgG
HRP-linked conjugate (Amersham Pharmacia Biotech) as secondary Ab at a
dilution of 1/5000 for 1 h at room temperature. Immunoreactive
bands were visualized by autoradiography using the ECL system (ECL+;
Amersham Pharmacia Biotech) according to the manufacturer’s
instructions.
Measurement of IL-1 and TNF- concentrations
The cell-free supernatants were analyzed for IL-1
and TNF-
using, respectively, human IL-1 and TNF- ELISA kits (Euroclone,
Wetherby, U.K.) according to the manufacturer’s instructions.
The lower limits of detection of these assays were 5 (IL-1) and 10
pg/ml (TNF-), respectively. In selected experiments, TNF-
bioactivity was measured by a cytotoxicity assay using the WEHI 164
clone 13 cell line, as described (48). IL-1 biological
activity was determined by the D10 G.4.1 proliferation assay
(49).
NF-B binding
Binding of NF-B p50 and p65 subunits to the NF-B binding
consensus sequence 5'-GGGACTTTCC-3' was measured with the ELISA-based
Trans-Am NF-B kit (Active Motif, Carlsbad, CA) using whole-cell
lysates prepared from monocyte monolayers. The Trans-Am kit employs
96-well microtiter plates coated with an oligonucleotide containing the
NF-B binding consensus sequence. The active forms of either the p50
or p65 subunits in whole-cell extracts can be detected using Abs
specific for an epitope that is accessible only when the subunit is
activated and bound to its target DNA. Preparation of cell extracts was
done exactly as recommended by the manufacturer. Specificity was
checked by measuring the ability of soluble wild-type or mutated
oligonucleotides to inhibit binding. Results are expressed as specific
binding, i.e., as the absorbance values observed in the presence of the
mutated oligonucleotide minus those observed in the presence of the
wild-type oligonucleotide. In preliminary experiments, the Trans-Am kit
showed a good correlation with an EMSA in detecting the DNA binding
capacity of NF-B.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is known that LPS stimulation leads to increased activation of
three subgroups of MAPKs, i.e., ERK, JNK, and p38, in murine
macrophages and human monocytes (38, 39). To investigate
whether these well-characterized pathways are also involved in GBS
signal transduction, human monocytes from adult donors were treated for
different periods of time with killed GBS or with LPS, used as a
positive control. Monocyte lysates were then examined for the activated
forms of MAPKs by Western blot analysis. As shown in Fig. 1
, all three MAPKs became phosphorylated,
and hence activated, in adult monocytes stimulated with GBS. ERK 1/2
and p38 activation was dose-dependent and maximal using 1–10 µg/ml
of lyophilized GBS. Similar results were observed in monocytes from
cord blood (data not shown).
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also shows that GBS-induced ERK 1/2 and p38 activation was
significantly delayed relative to that induced by LPS. Activation was
first detected at 30 min and peaked at 60 min after the addition of
GBS. In contrast, LPS-induced phosphorylation was clearly detectable at
15 min and peaked at 30 min (Fig. 1). Moreover, phosphorylation of the
JNK MAPK was similarly delayed in monocytes stimulated with GBS,
relative to those stimulated with LPS (data not shown). MAPK
activation went back to baseline levels at 120 and 180 min,
respectively, after LPS and GBS stimulation (Figs. 1 and 2). Therefore, MAPK activation appeared
not only to occur later, but also to persist for a longer time in
GBS-stimulated monocytes than in those stimulated with LPS.
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,
which are known to be induced by GBS (10). This was
indicated by the inability of neutralizing concentrations of IL-1ra or
anti-human TNF- Abs (10 and 1 µg/ml, respectively) to affect
GBS-mediated MAPK activation (Fig. 2). Moreover, endotoxin
contamination of GBS preparations was unlikely to account for MAPK
activation, as indicated by the inability of the endotoxin-inactivating
agent polymyxin B (20 µg/ml) to affect GBS-induced ERK, p38, or JNK
stimulation (data not shown).
Because MAPKs are known to be involved in activation phenomena leading
to cytokine production, it was of interest to ascertain whether the
observed delay in GBS-induced MAPK activation (Figs. 1 and 2) was
paralleled by a delay in TNF- secretion. Fig. 3 shows that this was the case, because
significant TNF- elevations were first detected at 1 and 2 h
after the application of LPS and GBS, respectively. Moreover,
GBS-induced TNF- release was also delayed relative to that induced
by the Gram-positive bacteria S. aureus and L.
monocytogenes (Fig. 3).
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secretion
To investigate whether MAPK activation plays a causal role in
TNF- production, selective inhibitors were used. The ERK pathway was
blocked by pretreatment with either PD98059, which blocks the
ERK-activating MAPKK MAP kinase kinase (MEK)1 (50),
or U0126, a novel agent which was initially considered as an inhibitor
of AP-1-driven gene transcription and later shown to directly
block MEK1 and MEK2 (51). In initial experiments, we
ensured that these inhibitors selectively blocked ERK activation. Fig. 4 shows that PD98059 (50 µM) completely
abrogated basal and GBS-stimulated ERK 1/2 but not p38 phosphorylation.
Fig. 4 also shows that the p38 inhibitor SB203580, a bicyclic imidazole
compound (52), selectively blocked p38 phosphorylation,
whereas the NF-B inhibitor CAPE (see below) did not affect MAPK
activation.
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was measured at different times after GBS (10 µg/ml)
stimulation of monocytes pretreated with the MAPK inhibitors PD98059 or
U0126. Both PD98059 and U0126, respectively, at 50 and 10 µM doses
significantly (albeit partially) reduced TNF- secretion at
all time points after stimulation (Fig. 5, A and B). As
shown in Fig. 5C, even greater inhibition of TNF- release
was produced by the specific p38 inhibitor SB203580. The latter, at a
20-µM concentration, totally prevented TNF- secretion during the
first 4 h after the application of GBS, although TNF- levels
were only partially reduced at 22 h. The addition of the DMSO
vehicle used to dissolve the inhibitors did not affect TNF-
production (Fig. 5).
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release. Monocytes were therefore treated with a combination of PD98059
and SB203580 before stimulation. Using a 25-µM concentration for each
inhibitor, the combined treatment resulted in the almost complete
reduction of immunologically detectable TNF- at 22 h after the
addition of GBS (Fig. 6A).
Similar results were observed using LPS, instead of GBS, as a stimulus
(Fig. 6B). At each of the concentrations studied, the
effects of the combined treatment were significantly higher than those
of either inhibitor alone (Fig. 6).
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B activation
In further studies it was of interest to investigate whether MAPK
phosphorylation had a causal role in the activation of NF-B, a
transcription factor which is thought to be involved in the expression
of proinflammatory cytokine genes. Therefore, the effects of ERK and
p38 blockade on NF-B activation were investigated. Human monocytes
were stimulated with GBS or LPS for different time intervals in the
presence or absence of inhibitors, and cytoplasmic extracts were probed
with phospho-IB-specific Ab. However, neither the ERK inhibitor
PD98059 nor the p38 inhibitor SB203580 affected IB
phosphorylation (Fig. 7, upper
panel). In the course of these studies it was noticed that
GBS-induced IB phosphorylation occurred within 60 min and was
maximal at 120 min. Again, these activation kinetics were considerably
delayed relative to LPS-induced activation, which was clearly
detectable at 15 min (Fig. 7, upper panel).
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B to DNA. Specifically, we looked at the
effects of the PD98059 and SB203580 inhibitors on binding of the p65
and p50 NF-B subunits to a consensus NF-B binding sequence. Fig. 7 (lower panel) shows that increased specific binding
of active p65 and p50 subunits was detected in monocyte lysates at 60
and 120 min after stimulation with GBS. However, neither the ERK
inhibitor PD98059 nor the p38 inhibitor SB203580 affected GBS-induced
NF-B binding (Fig. 7, lower panel). Similarly, these
inhibitors had no effect on NF-B binding when using LPS (1 µg/ml)
as a stimulus (data not shown).
Previous studies have shown that NF-B is activated in response to
GBS (15, 45), but the causal role of this phenomenon in
TNF- production is unclear. Therefore, human monocytes were
preincubated for 1 h with various concentrations of CAPE, a potent
NF-B inhibitor (53), before being challenged with GBS
(10 µg/ml) or LPS (1 µg/ml). As shown in Fig. 8, CAPE significantly inhibited the
production of TNF- induced by GBS. For example, a 49% inhibition
was observed with a 20-µM dose after 22 h.
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levels in monocytes pretreated with
CAPE-SB203580 and CAPE-PD98059 combinations. As shown in Fig. 9, both of these combinations almost
totally prevented TNF- release in GBS- or LPS-treated monocytes.
These effects were similar in adult and cord blood monocytes (data not
shown). These data indicated that the simultaneous blockade of NF-B
and either the p38 or the ERK 1/2 MAPKs was sufficient to completely
block TNF- responses to GBS.
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In mononuclear phagocytes, LPS triggers the activation of PKC,
which is thought to play an important role in signal transduction
(54, 55). Therefore, we studied the effects of PKC
inhibitors on GBS-induced TNF- production and on MAPK and NF-B
activation. Fig. 10 shows that Ro
31-8220 and calphostin C, at doses known to selectively inhibit PKC
(56), inhibited GBS- and LPS-induced TNF- release to a
similar extent, with inhibition values ranging from 60 to 70%.
GÖ 6976, which selectively blocks the
Ca2+-dependent PKC and PKCI isoenzymes
(57), also significantly reduced TNF- release in GBS-
and LPS-stimulated monocytes by 47 and 79%, respectively (Fig. 10).
Therefore, the inhibitory effects of GÖ 6976 were lower than
those induced by Ro 31-8220 and calphostin C in GBS- but not
LPS-stimulated monocytes. This may indicate a more prominent role for
Ca2+-independent PKC isoforms in GBS- but not
LPS-induced TNF- production. However, further studies are needed to
verify this possibility.
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shows that none of the PKC
inhibitors used affected either MAPK phosporylation or p50 binding to
target oligonucleotide sequences. IB phosphorylation and p65
binding were similarly unaffected by any of the PKC inhibitors used
(data not shown). These data indicated that PKC activation has a role
in TNF- response after stimulation with either GBS or LPS, although
these effects are mediated by NF-B- and MAPK-independent
mechanisms.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a central mediator of lethality in experimental GBS
disease (16). Therefore, elucidation of signal
transduction pathways involved in GBS-induced TNF- production may
lead to alternative strategies to treat sepsis caused by these
bacteria. Previous studies have demonstrated the essential role of MAPK
and NF-B activation in proinflammatory cytokine responses to a
number of microbial stimuli, including LPS (58),
mycoplasma membrane lipoproteins (40, 41), and
Staphylococcal peptidoglycan (59). However, little is
known of the role of MAPKs and NF-B pathways in GBS-induced cell
responses.
Our data indicate that GBS cause a transient and dose-dependent
activation of the MAPK family members ERK 1/2, p38, and JNK, as well as
NF-B activation in human monocytes. The availability of highly
selective ERK 1/2 and p38 inhibitors allowed us to directly assess the
role of these MAPKs in TNF- release.
Blockade of the p38 or ERK 1/2 pathways significantly, albeit
partially, reduced TNF- production after stimulation with GBS. These
data are in agreement with those of a recent study showing that a
different p38 inhibitor (SB202190) could significantly reduce
GBS-induced TNF- production, as well as activation of the AP-1
transcription factor (45). However, the ERK MAPK pathway
was not examined in that study. Interestingly, in the present study,
combined treatment with ERK and p38 inhibitors had additive effects and
totally abrogated TNF- production. The mechanism(s) underlying
these effects are presently unclear. The simultaneous activation of
multiple MAPKs may be required to enhance transcription by a mechanism
involving interaction of the transcriptional initiation site of the TNF
promoter with RNA polymerase II (37) and/or to increase
mRNA stability and translational efficiency (60, 61).
MAPKs may also increase TNF transcription via activation of a number of
specific transcription factors, including AP-1 and NF-B. AP-1
activation was prevented by a p38 MAPK inhibitor in GBS-stimulated
neonatal monocytes (45). Moreover, in the present study,
the involvement of NF-B in GBS-induced TNF- production was
documented using CAPE, a selective translocation inhibitor. Thus, it
was of interest to ascertain whether MAPK activation had a causal role
in IB phosphorylation, and hence NF-B activation, or in
NF-B binding to target DNA sequences, as previously shown in other
experimental systems (36, 62, 63). However, this was not
the case in GBS-stimulated monocytes, suggesting that the MAPK and
NF-B pathways are independently activated.
Similarly, divergence in these signaling pathways was observed with a
selective Src tyrosine kinase inhibitor, which blocked LPS-induced ERK
and p38 MAPK activation, as well as TNF- production, but had no
effect on IB degradation or NF-B binding (64). It
cannot be excluded, however, that the MAPKs and NF-B pathways
converge further down in the activation cascade. This is actually
suggested by studies in which MAPK blockade prevented NF-B-dependent
gene transcription without affecting IB phosphorylation or
NF-B binding (65, 66, 67).
Because PKC was previously shown to have an important role in cytokine
responses to bacterial stimuli (54, 55), we investigated
whether PKC inhibitors blocked MAPK or NF-B activation. However,
this was not the case, although PKC inhibitors did significantly reduce
both LPS- and GBS-induced TNF- release.
Collectively, our data indicate that the strong cytokine response
observed with GBS requires the independent activation of several
pathways, including the MAPK (ERK and p38) and NF-B pathways.
Accordingly, complete inhibition of TNF- release appears to require
blockade of more than one intracellular mediator. Therefore, future
therapeutic strategies aimed at preventing TNF- production could
perhaps exploit the simultaneous blockade of multiple signaling events
using combined treatments.
Alternatively, complete inhibition of TNF- release may be achieved
by blocking an upstream reaction leading to both MAPK and NF-B
activation. In fact, recent data indicate that MAPKs and NF-B can be
activated by a common upstream signal transduction pathway
(68). This pathway is shared by all members of the TLR and
IL-1R families and includes the adaptor protein MyD88, the
IL-1R-associated kinase, and TNFR-associated factor 6
(68). Interestingly, a recent study indicates that MyD88
is essential in GBS-induced cytokine responses, as shown in mice with a
targeted disruption of this adaptor protein (31)
In the present study, protein phosphorylation and the effects of
specific inhibitors were assessed in parallel using LPS or GBS as
stimuli. Very similar results were obtained, indicating that MAPKs and
NF-B play similar roles in GBS- and LPS-induced monocyte activation.
This may be clinically relevant because similar NF-B or MAPK
inhibitors, alone or in combination, could potentially be used
therapeutically to prevent septic shock during infections caused by GBS
and Gram-negative bacteria. Recently, the p38 kinase inhibitor SB203580
has been shown to markedly improve survival, even when administered as
a delayed posttreatment to mice subjected to polymicrobial sepsis by
cecal ligation puncture (69).
Differences between GBS- and LPS-induced activation were also
documented here. Using GBS as a stimulus, ERK, p38, and IB
phosporylation, as well as TNF- production, were significantly
delayed (30–60 min), relative to LPS. This phenomenon was not related
to weaker monocyte stimulation by GBS because, relative to LPS, GBS
induced higher maximal TNF- levels (Fig. 2). Rather, differences in
cell activation kinetics may reflect differences in the nature of early
signal transduction mechanisms, including the activating receptors.
This possibility is suggested by observations that GBS can fully
activate macrophages from LPS-hyporesponsive mice (15).
Moreover, GBS activate human and rodent cells by mechanisms that do not
involve CD14 (13, 15, 70), a well-known LPS coreceptor.
The nature of the activating GBS receptor is under active
investigation. TLR2, which can mediate responses to other Gram-positive
bacteria such as L. monocytogenes, is apparently not
involved in GBS-induced stimulation (30). Interestingly,
GBS-induced cell activation is delayed relative to that induced not
only by LPS, but also by L. monocytogenes and other
Gram-positive bacteria (Fig. 2).
In conclusion, it is shown here that the simultaneous activation of
multiple pathways, including NF-B, ERK 1/2, and p38 MAPK, is
required to induce maximal TNF- production. Accordingly, in the
clinical setting, complete inhibition of TNF- release may require
treatment with single drugs or drug combinations capable of inhibiting
multiple activation pathways.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Giuseppe Teti, Department of Experimental Pathology and Experimental Microbiology, Torre Biologica (IIp.) Policlinico Universitario Via Consolare Valeria, 1 (Gazzi) I-98125 Messina, Italy. E-mail address: teti{at}eniware.it
3 Abbreviations used in this paper: GBS, group B streptococcus; TLR, Toll-like receptor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPKK, MAPK kinase; CAPE, caffeic acid phenetyl ester; IL-1ra, IL-1R antagonist; MEK, MAP kinase kinase; PKC, protein kinase C.
Received for publication September 11, 2001. Accepted for publication May 3, 2002.
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  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]
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 G. Mancuso, A. Midiri, C. Biondo, C. Beninati, S. Zummo, R. Galbo, F. Tomasello, M. Gambuzza, G. Macri, A. Ruggeri, et al. Type I IFN Signaling Is Crucial for Host Resistance against Different Species of Pathogenic Bacteria J. Immunol., March 1, 2007; 178(5): 3126 - 3133. [Abstract] [Full Text] [PDF]
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 I. Venza, M. Cucinotta, S. Caristi, G. Mancuso, and D. Teti Transcriptional Regulation of IL-8 by Staphylococcus aureus in Human Conjunctival Cells Involves Activation of AP-1 Invest. Ophthalmol. Vis. Sci., January 1, 2007; 48(1): 270 - 276. [Abstract] [Full Text] [PDF]
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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]
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S.-B. Lee and J. S. Schorey Activation and Mitogen-Activated Protein Kinase Regulation of Transcription Factors Ets and NF-{kappa}B in Mycobacterium-Infected Macrophages and Role of These Factors in Tumor Necrosis Factor Alpha and Nitric Oxide Synthase 2 Promoter Function Infect. Immun., October 1, 2005; 73(10): 6499 - 6507. [Abstract] [Full Text] [PDF]
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G. Mancuso, A. Midiri, C. Biondo, C. Beninati, M. Gambuzza, D. Macri, A. Bellantoni, A. Weintraub, T. Espevik, and G. Teti Bacteroides fragilis-Derived Lipopolysaccharide Produces Cell Activation and Lethal Toxicity via Toll-Like Receptor 4 Infect. Immun., September 1, 2005; 73(9): 5620 - 5627. [Abstract] [Full Text] [PDF]
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 S. K. Selvaraj and N. V. Prasadarao Escherichia coli K1 inhibits proinflammatory cytokine induction in monocytes by preventing NF-{kappa}B activation J. Leukoc. Biol., August 1, 2005; 78(2): 544 - 554. [Abstract] [Full Text] [PDF]
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S. Caristi, G. Piraino, M. Cucinotta, A. Valenti, S. Loddo, and D. Teti Prostaglandin E2 Induces Interleukin-8 Gene Transcription by Activating C/EBP Homologous Protein in Human T Lymphocytes J. Biol. Chem., April 15, 2005; 280(15): 14433 - 14442. [Abstract] [Full Text] [PDF]
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M. K. Dahle, G. Overland, A. E. Myhre, J. F. Stuestol, T. Hartung, C. D. Krohn, O. Mathiesen, J. E. Wang, and A. O. Aasen The Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway Is Activated by Lipoteichoic Acid and Plays a Role in Kupffer Cell Production of Interleukin-6 (IL-6) and IL-10 Infect. Immun., October 1, 2004; 72(10): 5704 - 5711. [Abstract] [Full Text] [PDF]
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H. Mikamo, A. K. Johri, L. C. Paoletti, L. C. Madoff, and A. B. Onderdonk Adherence to, Invasion by, and Cytokine Production in Response to Serotype VIII Group B Streptococci Infect. Immun., August 1, 2004; 72(8): 4716 - 4722. [Abstract] [Full Text] [PDF]
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G. Mancuso, A. Midiri, C. Beninati, C. Biondo, R. Galbo, S. Akira, P. Henneke, D. Golenbock, and G. Teti Dual Role of TLR2 and Myeloid Differentiation Factor 88 in a Mouse Model of Invasive Group B Streptococcal Disease J. Immunol., May 15, 2004; 172(10): 6324 - 6329. [Abstract] [Full Text] [PDF]
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A. Bafica, C. A. Scanga, O. Equils, and A. Sher The induction of Toll-like receptor tolerance enhances rather than suppresses HIV-1 gene expression in transgenic mice J. Leukoc. Biol., March 1, 2004; 75(3): 460 - 466. [Abstract] [Full Text] [PDF]
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M. Galdiero, M. Galdiero, E. Finamore, F. Rossano, M. Gambuzza, M. R. Catania, G. Teti, A. Midiri, and G. Mancuso Haemophilus influenzae Porin Induces Toll-Like Receptor 2-Mediated Cytokine Production in Human Monocytes and Mouse Macrophages Infect. Immun., February 1, 2004; 72(2): 1204 - 1209. [Abstract] [Full Text] [PDF]
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V. Cusumano, A. Midiri, V. V. Cusumano, A. Bellantoni, G. De Sossi, G. Teti, C. Beninati, and G. Mancuso Interleukin-18 Is an Essential Element in Host Resistance to Experimental Group B Streptococcal Disease in Neonates Infect. Immun., January 1, 2004; 72(1): 295 - 300. [Abstract] [Full Text] [PDF]
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K. Gee, M. Kozlowski, and A. Kumar Tumor Necrosis Factor-{alpha} Induces Functionally Active Hyaluronan-adhesive CD44 by Activating Sialidase through p38 Mitogen-activated Protein Kinase in Lipopolysaccharide-stimulated Human Monocytic Cells J. Biol. Chem., September 26, 2003; 278(39): 37275 - 37287. [Abstract] [Full Text] [PDF]
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S.-W. Yoon, S.-H. Goh, J.-S. Chun, E.-W. Cho, M.-K. Lee, K.-L. Kim, J.-J. Kim, C.-J. Kim, and H. Poo {alpha}-Melanocyte-stimulating Hormone Inhibits Lipopolysaccharide-induced Tumor Necrosis Factor-{alpha} Production in Leukocytes by Modulating Protein Kinase A, p38 Kinase, and Nuclear Factor {kappa}B Signaling Pathways J. Biol. Chem., August 29, 2003; 278(35): 32914 - 32920. [Abstract] [Full Text] [PDF]
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