or IL-1ß on cellular signaling and mediator production
* Departments of Physiology and Neuroscience and
Pharmacology and Medicine, Medical University of South Carolina, Charleston, and
Institute of Pharmacology, Medical University of Messina, Italy
Correspondence: Prof. James A. Cook, Dept. of Physiology and Neuroscience, 167 Ashley Ave., Suite 617 Storm Eye Institute, Charleston, SC 29425. E-mail: cookja{at}musc.edu
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and interleukin (IL)-1ß.
Post-receptor convergence of signal transduction pathways might occur
after LPS, IL-1ß, and TNF- stimulation. Therefore, it was
hypothesized that down-regulation of common signaling molecules induces
cross-tolerance among these stimuli. LPS tolerance and cross-tolerance
were examined in THP-1 cells. Phosphorylation of MAP kinases and
degradation of inhibitor 
B (IB) DNA binding of nuclear
factor-B (NF-B), and mediator production were examined. In naive
cells, LPS, TNF-, and IL-1ß induced IB degradation, kinase
phosphorylation, and NF-B DNA binding. LPS stimulation induced
production of TNF- or TxB2 and degradation of IRAK.
However, neither TNF- nor IL-1ß induced IRAK degradation or
stimulated TNF- or TxB2 production in naive cells.
Pretreatment with each stimulus induced homologous tolerance to
restimulation with the same agonist. LPS tolerance also suppressed
LPS-induced TxB2 and TNF- production. LPS
pretreatment induced cross-tolerance to TNF- or IL-1ß stimulation.
Pretreatment with TNF- induced cross-tolerance to LPS-induced
signaling events and TxB2 production. Although pretreatment
with IL-1ß did not induce cross-tolerance to LPS-induced signaling
events, it strongly inhibited LPS TNF- and TxB2
production. These data demonstrate that IL-1ß induces cross-tolerance
to LPS-induced mediator production without suppressing LPS-induced
signaling to MAP kinases or NF-B activation.
Key Words: macrophage/monocyte • signal transduction • phosphorylation • MAP kinases
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(TNF-), and arachidonic acid metabolites from various
cell types, but predominantly by macrophages and monocytes
[1
].
LPS induction of pro- and anti-inflammatory molecules is mediated
through activation of the glycosylphosphatidylinositol-linked protein
CD14 receptor [2
], Toll-like receptor (TLR) 4
[3
, 4
], and intracellular signaling
pathways. Through a series of post-receptor molecules, LPS induces
phosphorylation and degradation of inhibitor protein B (IB)
, which inhibits nuclear translocation of transcription nuclear factor
(NF) B (NF-B) [5
]. LPS also activates the
mitogen-activated protein (MAP) kinases, including extracellular
regulated kinase (ERK)1/2 [6
], p38 kinase
[7
, 8
] and c-Jun N-terminal kinase (JNK)
[9
].
LPS is able to induce tolerance or desensitization to subsequent LPS
challenge. Prior exposure to low concentrations of LPS in vitro or
low-dose LPS challenge in vivo suppresses macrophage/monocyte
proinflammatory mediator production and improves survival to otherwise
lethal LPS shock [10
]. Although LPS tolerance does not
induce global suppression of mediators, a reduction in
proinflammatory-cytokine production, e.g., TNF- and thromboxane (Tx)
B2, is observed during tolerance [11
]. The
decreased mediator release has been linked to altered signal
transduction pathways. In tolerance, down-regulation of MAP kinases
[6
, 8
] and alterations of NF-B DNA
binding have been reported [12
]. LPS tolerance can also
confer cross-tolerance to a variety of other noxious stimuli, e.g.,
certain gram-positive bacteria [13
] and ischemia
reperfusion injury [14
]. Cross-tolerance of LPS to
TNF- or IL-1ß also has been demonstrated by other investigators
[15
16
17
18
19
].
Recently, it has been reported that down-regulation of TLR4
expression occurs in murine peritoneal macrophages, which may be one of
the molecular mechanisms of LPS tolerance [20
]. Proteins
that belong to the IL-1ß signal transduction pathway, such as myeloid
differentiation factor 88 (MyD88), IL-1 receptor associated kinase
(IRAK), TNF receptor-activated factor 6 and NF-B-inducing kinase,
are coupled to TLR4 and are involved in LPS activation of NF-B and
MAP kinases [21
22
23
]. Also the TNF- signal
transduction pathway may converge, through activation of TRAF2, at the
level of NF-B-inducing kinase leading to nuclear translocation of
NF-B [24
, 25
].
Because potential post-receptor convergent signal transduction pathways
are activated in response to LPS, IL-1ß, and TNF-, we hypothesized
that down-regulation of this pathway is a primary mechanism for
cross-tolerance between LPS and TNF- or IL-1ß. Therefore, the aim
of this study was to evaluate whether these inflammatory cytokines can
induce cross-tolerance to LPS and vice versa in human promonocytic
THP-1 cells. Specifically alterations of signal transduction pathways
leading to degradation of IRAK and IB, or nuclear translocation
of NF-B, activation of MAP kinases and production of TNF- and
TxB2 were examined.
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Tolerance to LPS, TNF-, and IL-1ß in human promonocyte THP-1 cells
was examined. In the first series of experiments, the cells were
rendered LPS tolerant by pretreatment with different LPS concentrations
(10 ng/mL, 100 ng/mL, and 1 µg/mL) for 18 h and subsequently
stimulated with LPS (10 µg/mL), TNF- (10 ng/mL), or IL-1ß (100
ng/mL) for 40 min. Tolerance was also induced by pretreatment with LPS
(1 µg/mL), TNF- (10 ng/mL), or IL-1ß (100 ng/mL) for 18 h,
and subsequent stimulation was with LPS (10 µg/mL), TNF- (10
ng/mL), or IL-1ß (100 ng/mL) for 40 min, 2 h, or 18 h. ERK
and JNK phosphorylation, IRAK and IB degradation, NF-B DNA
binding, and TNF- or TxB2 production were examined.
Cell lysate preparation
After appropriate stimulation, cells were centrifuged, and the
pellet was kept on ice-cold RIPA lysis buffer [20 mM HEPES, pH 7.4,
1% Triton X-100, 50 mM NaCl, 1 mM EGTA, 5 mM ß-glycerophosphate, 30
mM sodium pyrophosphate, 100 mM sodium orthovanadate, 0.1 mM
phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL of leupeptin, and 10
µg/mL of pepstatin A) for 15 min, sonicated for a few seconds, and
then centrifuged for 10 min at 4°C at 10,000 g. An aliquot
was taken for protein determination, and the remaining supernatant was
stored at -20°C until Western blot analysis was performed.
Western blot analysis
For detection of ERK, JNK, IRAK, and IB, cellular lysates
were added to Laemmli sample buffer [26
] and boiled for
5 min. Subsequently, protein from each sample was loaded onto a sodium
dodecyl sulfate-4–12% polyacrylamide gel, subjected to
electrophoresis, and transferred onto a PVDF membrane. For
immunodetection, membranes were washed with Tris-buffered saline-Tween
20 (TBS-T; 20 mM Tris, 500 mM NaCl, 0.1% Tween 20) and blocked in a
7% powdered-milk solution in TBS-T for 1 h. After three washes
with TBS-T, membranes were incubated for 1 h or overnight with
either one of the following polyclonal antibodies in TBS-T: anti-JNK,
specific for the dual phosphorylated form on Thr183/tyr185 of JNK
(1:1,000) (Promega, Madison, WI); anti-ERK specific for the dual
phosphorylated form on Thr202/Tyr204 (1:1,000) (New England Biolabs,
Inc., Beverly, MA); anti-IB (1:1000) (Cell Signaling, Beverly,
MA); or anti-IRAK, recognizing IRAK at 80 kDa (1:1,000) (Upstate, Lake
Placid, NY). Membranes were washed again before being incubated with a
horseradish peroxidase-conjugated donkey anti rabbit-immunoglobulin
(Ig) G antibody (1:4,000) (Amersham Pharmacia Biotech, Inc.,
Piscataway, NJ) in blocking buffer for 1 h. After the last three
washes with TBS-T (TBS-0.15% Tween-20 ), protein detection was
visualized by incubation with ECL Plus detection reagents (Amersham
Pharmacia Biotech, Inc.) for 5 min and development of the exposed
enhanced-chemoluminescence hyperfilms (Amersham Pharmacia Biotech,
Inc.).
Nuclear extraction
Nuclear extracts were isolated by a modified procedure of
Dignam et al. [27
]. Briefly, after treatment, cells were
spun at 2,800 g for 10 min. The cell pellet was resuspended
in 400 µL of hypotonic buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1
mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM PMSF, and 10
µg/mL of the following protease inhibitors: leupeptin, aprotinin, and
pepstatin], set on ice for 10 min, and vortexed for 4 s with
0.6% Nonidet-P40. After centrifugation at 2,800 g for 10
min at 4°C, the pellet was resuspended in 15 µL of buffer C (20 mM
HEPES, pH 7.9, 1 M NaCl, 5% glycerol, 1 mM EDTA, 1 mM EGTA, 1 mM DTT,
0.5 mM PMSF, 10 µg/mL of protease inhibitor) and gently agitated on
an orbital shaker at 4°C for 30 min. After centrifugation at 14,000
g for 10 min, an aliquot of each supernatant was taken for
protein determination, and the remaining supernatant was stored at
-70°C.
Electrophoretic mobility shift assay
Double-stranded consensus binding site oligonucleotide for
NF-B was obtained from Promega and was end-labeled with
[
-32P]ATP (NEN Life Science Products, Boston, MA)
using T4 polynucleotide kinase (Promega, Madison, WI). For binding
reactions, 10 µg of nuclear extracts were incubated in 20 µL of
total reaction mix (20 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, 5%
glycerol, 1 mM DTT, 250 µg/mL of bovine serum albumin) containing the
nonspecific competitors pd(N)6 and poly(dI-dC)-poly(dI-dC)
(Amersham Pharmacia Biotech). The labeled oligonucleotide was added,
and the mixture was incubated for 20 min at room temperature. Samples
were analyzed by 4% nondenaturing polyacrylamide gel electrophoresis,
and the gel was dried and exposed to Hyperfilm ECL at -70°C.
Antibodies against p50 and p65 (Santa Cruz Biotechnology Inc., Santa
Cruz, CA) subunits of NF-B were used for the supershift analysis.
TNF- assay
TNF- was measured using an enzyme-linked immunosorbant assay.
Briefly, anti-human TNF- antibody (0.8 µg/mL) (R&D System Inc.
Minneapolis, MN) was used to coat 96-well enhanced-binding plates
overnight at 4°C. The plates were washed with PBS/Tween-20 and
blocked for 2 h with blocking buffer containing 10% bovine serum
albumin. Plates were washed, and standards and samples were added and
incubated overnight at 4°C. After this washing, biotinylated
anti-human TNF- antibody (0.3 µg/mL) ((R&D System Inc.) was added
to the wells, and plates were incubated for 1 h at room
temperature. Plates were washed, streptavidin-peroxidase conjugate
(1:2,000) (BioSourse International Inc., Camarillo, CA) was added, and
plates were incubated for 45 min. Finally, after washing, the ABTS
substrate solution containing 3% H2O2 was
added, and plates were read at 415 nm within 1 h.
TxB2 assay
After treatment, cells were centrifuged, and supernatants were
stored at -20°C. Samples were diluted 1:10 in buffer containing
0.1% polyvinylpyrolidine, 0.9% NaCl, 50 mM Tris base, 1.7 mM
MgSO4, and 0.16 mM CaCl2 (pH 7.4) prior to
radioimmunoassay. TxB2 was quantitated by radioimmunoassay
as previously described [28
, 29
].
Data analysis
Gels were analyzed by scanning densitometry, and analysis was
performed on a Macintosh computer using the public domain National
Institutes of Health (NIH; Bethesda, MD) Image program (available on
the Internet at http://rsb.info.nih.gov/nih-image/). Statistical
significance was determined by one-way analysis of variance with
Fischer’s post-hoc correction using Statview software. A
P value of <0.05 was considered significant. Data are
expressed as means ± SE.
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B degradation in naive and tolerant cells (10
ng/mL), or IL-1ß (100 ng/mL) for 40 min. Whereas LPS, TNF-, and
IL-1ß stimulation induced evident IB degradation in naive cells
(i.e., cells not pretreated with LPS), in LPS-pretreated cells with
subsequent LPS, TNF-, and IL-1ß, induced degradation of IB
was suppressed (Fig. 1a
1b
1c
).
 View larger version (47K): [in a new window] |
Figure 1. Effect of pretreatment with different LPS concentrations on IB
cellular content. THP-1 cells were pretreated (pretr.) with medium
alone or LPS (10 ng/mL, 100 ng/mL, and 1000 ng/mL) for 18 h and
then stimulated (stim.) with LPS (10 µg/mL) (a), TNF- (10 ng/mL)
(b), or IL-1ß (100 ng/mL) (c) for 40 min. Western blot analysis was
carried out to detect IB cellular content as described in
Materials and Methods. Results are representative of three independent
experiments.
|
B was quantitatively determined by scanning
densitometry after stimulation of THP-1 cells with LPS (10 µg/mL),
TNF- (10 ng/mL), or IL-1ß (100 ng/mL) for 40 min. A reduction
(n =3; P<0.05) in cellular content of
IB was induced by treatment with LPS (93.1±4.2%), TNF-
(94.1±3.5%), and IL-1ß (75.1±8.1%) compared with basal
(Fig. 2a
).
 View larger version (45K): [in a new window] |
Figure 2. IB cellular content in LPS-, TNF--, or IL-1ß-pretreated
cells. (a) THP-1 cells were incubated with LPS (10 µg/mL), TNF-
(10 ng/mL), IL-1ß (100 ng/mL), or medium alone (basal) for 40 min.
(b) Cells were rendered LPS tolerant by pretreatment for 18 h with
LPS (1 µg/mL) and subsequently stimulated with LPS (10 µg /mL),
TNF- (10 ng/mL), IL-1ß (100 ng/mL), or medium alone (basal) for 40
min. (c) Cells were rendered TNF- tolerant by pretreatment with
TNF- (100 ng/mL) for 18 h and then stimulated with LPS (10
µg/mL), TNF- (10 ng/mL), or medium alone (basal) for 40 min. (d)
Cells were rendered IL-1ß tolerant by pretreatment with IL-1ß (100
ng/mL) for 18 h and subsequently stimulated with LPS (10 µg
/mL), IL-1ß (100 ng/mL), or medium alone (basal) for 40 min. Detected
proteins were quantified by scanning densitometry, and results were
expressed as percentages of basal value, which was arbitrarily assigned
100%. Data represent the mean ± SE from three to
four independent experiments. *, P < 0.05 compared with
basal of each group; #, P < 0.05 compared with stimulation
in naive group.
|
(10 ng/mL), or
IL-1ß (100 ng/mL) for 40 min (Fig. 2b)
. Although some reduction in
IB cellular content was observed after TNF-
(36.1±5.4%), LPS (39.5±9.2%) or IL-1ß (28.1±1.7%) stimulation,
there was a significant decrease (n=3–4;
P<0.05) in degradation of IB induced by these stimuli
in tolerant cells compared with that in naive cells.
THP-1 cells were rendered TNF- tolerant by pretreatment with TNF-
for 18 h followed by stimulation with either LPS (10 µg/mL) or
TNF- (10 ng/mL) for 40 min, and the IB content was measured.
Stimulation with TNF- resulted in only marginal degradation of
IB (14.1±17%) which was significantly different
(P<0.05) from that observed in naive cells (Fig. 2c)
. In
contrast, LPS stimulation in TNF--tolerant cells induced IB
degradation similarly to that in naive cells.
When cells were pretreated with IL-1ß (Fig. 2d)
and subsequently
stimulated with LPS or IL-1ß, a pattern similar to the ones observed
in TNF--pretreated cells was induced. IB was degraded by LPS
stimulation, whereas in IL-1ß-pretreated cells, stimulation with
IL-1ß did not result in degradation of IB.
NF-B DNA binding
Because degradation of IB leads to nuclear translocation of
NF-B, we characterized NF-B DNA binding using antibodies against
the predominant proteins comprising NF-B, i.e., RelA (or p65) and
NF-B1 (or p50). A concentration-dependent increase in NF-B DNA
binding was observed when cells were stimulated with LPS (0.01–10
µg/mL) (Fig. 3
). Supershifts with antibody to the p50 or p65 subunits
demonstrated that these NF-B subunits were activated by LPS.
 View larger version (37K): [in a new window] |
Figure 3. Characterization of NF-B DNA binding. A dose-response to LPS was
examined by stimulating THP-1 cells with LPS (0.01 µg/mL to 10
µg/mL for 40 min). Electrophoretic mobility shift assay was performed
as described in Materials and Methods. Characterization of NF-B DNA
binding was evaluated using anti-p50 and anti-p65 antibodies. The
arrows show the supershift induced by anti-p50 and -p65 antibodies;
n.s., nonspecific binding. Free probe is not shown.
|
, and IL-1ß induced p65/p50-DNA binding
in naive THP-1 cells (Fig. 4
). LPS and TNF- pretreatment for 18 h induced a substantial
increase in p50/p50 homodimer-DNA binding which was not altered after
stimulation with LPS, TNF-, or IL-1ß. In contrast, IL-1ß
pretreated cells did not show an increase in basal NF-B-DNA binding,
and LPS-induced DNA binding was comparable with that in naive cells
although stimulation with IL-1ß did not result in DNA binding.
 View larger version (51K): [in a new window] |
Figure 4. NF-B DNA binding in naive and tolerant cells. THP-1 cells were
pretreated (pretr.) with medium alone (naive) or LPS (1 µg /mL),
TNF- (10 ng/mL), or IL-1ß (100 ng/mL) for 18 h and
subsequently stimulated (stim) as is shown, for 40 min with LPS (10
µg/mL), TNF- (10 ng/mL), or IL-1ß (100 ng/mL). Electrophoretic
mobility shift assay was carried out as described in Materials and
Methods. The electrophoretic mobility shift assay shown is
representative of three independent experiments.
|
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(10
ng/mL), or IL-1ß (100 ng/mL) for 40 min. All three stimuli induced
evident phosphorylation of ERK in naive cells, which was suppressed in
LPS-tolerant groups in a concentration-dependent manner (Fig. 5a
5b
5c
).
 View larger version (58K): [in a new window] |
Figure 5. Effect of pretreatment with different LPS concentrations on ERK
phosphorylation. THP-1 cells were pretreated (pretr.) with medium alone
(naive) or LPS (10 ng/mL, 100 ng/mL, and 1000 ng/mL) for 18 h and
then stimulated (stim.) with LPS (10 µg/mL) (a), TNF- (10 ng/mL)
(b), or IL-1ß (100 ng/mL) (c) for 40 min. Results are representative
of three independent experiments for each group.
|
(10
ng/mL), or IL-1ß (100 ng/mL) pretreatment followed by stimulation
with LPS, TNF-, or IL-1ß on ERK activation was determined. ERK was
induced in naive cells after stimulation with all stimuli. When cells
were rendered LPS- , TNF-- , or IL-1ß-tolerant, no phosphorylation
of ERK was observed after stimulation with the same agonist. LPS- and
TNF--induced reciprocal cross-tolerance as evidenced by suppressed
ERK phosphorylation (Fig. 6
). In contrast, in IL-1ß-tolerant cells, ERK was activated by
stimulation with LPS.
 View larger version (8K): [in a new window] |
Figure 6. ERK phosphorylation in LPS-, TNF--, or IL-1ß-pretreated cells.
Cells were pretreated (pretr.) with LPS (1 µg/mL), TNF- (10
ng/mL), IL-1ß (100 ng/mL), or medium alone (naive) for 18 h and
subsequently stimulated (stim) as shown for 40 min with LPS (10
µg/mL), TNF- (10 ng/mL), or IL-1ß (100 ng/mL). Western blot was
carried out as described in Materials and Methods. Results are
representative of two independent experiments.
|
, or IL-1ß. All of these stimuli induced strong
phosphorylation of JNK in naive cells, which was suppressed in
LPS-tolerant groups in a concentration-dependent manner (Fig. 7a
7b
7c
).
 View larger version (52K): [in a new window] |
Figure 7. Effect of pretreatment with different LPS concentrations on JNK
phosphorylation. THP-1 cells were pretreated (pretr.) with medium alone
(naive) or LPS (10 ng/mL, 100 ng/mL, and 1000 ng/mL) for 18 h and
then stimulated (stim.) with LPS (10 µg/mL) (a), TNF- (10 ng/mL)
(b), or IL-1ß (100 ng/mL) (c) for 40 min. Results are representative
of three independent experiments for each group.
|
(10
ng/mL), or IL-1ß (100 ng/mL) pretreatment followed by stimulation
with LPS, TNF-, or IL-1ß on JNK activation was determined. LPS,
TNF-, and IL-1ß stimulation induced JNK phosphorylation in
comparison with naive basal level (Fig. 8
). However the degree of activation with IL-1ß and TNF- was
less than with LPS. In LPS-, TNF--, and IL-1ß-tolerant cells,
basal levels of JNK phosphorylation were significantly higher than in
naive cells. The higher basal naive activation observed in the tolerant
groups probably reflects some degree of residual activation of JNK
after tolerance induction. However there was no further increase in JNK
phosphorylation after stimulation with LPS or the cytokines. Although
LPS-tolerant cells did not show an increase in JNK phosphorylation
after LPS stimulation, in TNF-- and IL-1ß-tolerant cells,
LPS-induced JNK phosphorylation was not impaired.
 View larger version (28K): [in a new window] |
Figure 8. JNK phosphorylation in LPS-, TNF--, or IL-1ß-pretreated cells. JNK
(46- and 54-kDa proteins) phosphorylation was in THP-1 cells. Cells
were pretreated (pretr.) with LPS (1 µg/mL), TNF- (10 ng/mL),
IL-1ß (100 ng/mL), or medium alone (naive) for 18 h and
subsequently stimulated (stim.) for 40 min with LPS (10 µg/mL),
TNF- (10 ng/mL), or IL-1ß (100 ng/mL). Results are representative
of two experiments.
|
, or IL-1ß for 40
min or 2 or 18 h, and cellular content of IRAK was observed. After
40 min of stimulation by all stimuli, no changes in IRAK cellular
content were observed (Fig. 9a
). LPS stimulation induced IRAK degradation at 2 h, which
persisted to 18 h. Stimulation with TNF- or IL-1ß did not
induce changes in cellular content of IRAK. However, when cells were
rendered TNF- or IL-1ß tolerant, the following LPS stimulation for
2 h induced IRAK degradation as in naive cells (Fig. 9b)
.
 View larger version (44K): [in a new window] |
Figure 9. IRAK cellular content. (a) THP-1 cells were stimulated (stim.) with LPS
(10 µg/mL), IL-1ß (100 ng/mL), or TNF- (10 ng/mL) for 40 min,
2 h, or 18 h. (b) Cells were pretreated (pretr.) with IL-1ß
(100 ng/mL) or TNF- (10 ng/mL) for 18 h and subsequently
stimulated for 2 h with LPS (10 µg/mL).
|
(supernatant release)
production. LPS pretreatment induced a concentration-dependent decrease
in TNF- production (Fig. 10
).
 View larger version (18K): [in a new window] |
Figure 10. Effect of pretreatment with different concentrations of LPS on TNF-
release in naive and tolerant cells. THP-1 cells were rendered LPS
tolerant by pretreatment with LPS (1, 10, or 100 ng/mL or 1000 ng/mL)
or medium alone (naive) for 18 h and then stimulated with LPS (10
µg/mL) for another 18 h. Data represent the means ±
SE from three independent experiments. *, P <
0.05 compared with basal (naive group); #, P < 0.05
compared with LPS stimulation in naive group.
|
production was
evaluated (Fig. 11
). LPS (10 µg/mL) induced a significant TNF- production
[2,016±337 pg/mL (n=3)] compared with basal [23±7 pg/mL
(n=3)], whereas IL-1ß did not induce TNF-.
LPS-pretreated cells showed a classic pattern of tolerance, as LPS
stimulation failed to induce TNF- production [172±39 pg/mL
(n=3)]. IL-1ß did not stimulate TNF- production in
naive cells. It is important, however, that IL-1ß pretreatment
induced a cross-tolerance to LPS-stimulated TNF- production. Indeed,
a significant decrease [P<0.05 (n=3)] in
TNF- production was observed after LPS stimulation [480±152 pg/mL
(n=3)] in IL-1ß-pretreated cells compared with naive
cells [2,016±337 pg/mL (n=3)].
 View larger version (17K): [in a new window] |
Figure 11. TNF- release in LPS, TNF- or IL-1ß pretreated cells. TNF-
production was evaluated in the supernatant of naive, LPS- or
IL-1ß-tolerant THP-1 cells. Cells were pretreated (pretr.) with
medium alone (naive), LPS (1 µg/mL), or IL-1ß (100 ng/mL) for
18 h and subsequently stimulated (stim.) with medium alone
(basal), LPS (10 µg/mL), and IL-1ß (100 ng/mL) for 18 h. Data
represent the means ± SE from three independent
experiments. *, P < 0.05 compared with basal (naive) group;
#, P < 0.05 compared with naive LPS stimulation.
|
begins with a long amino acid propeptide
sequence which is transferred to the plasma membrane as a trimeric
protein [30
]. TNF- is released in its mature form
from the plasma membrane by enzymatic cleavage [31
].
Because release of TNF- is subject to this complex process, we
evaluated whether in LPS- or IL-1ß-tolerant cells an alteration of
this process could occur. Cell-associated TNF- was quantitated in
tolerant cells after LPS or IL-1ß restimulation. As shown by
Figure 12
, cellular concentrations of TNF- were similar to ones measured
in the supernatant of the corresponding groups. LPS induced a
significant increase (P<0.05) in cell-associated TNF-
[2,143±556 pg/mL (n=3)] in naive cells compared with
basal (62±3pg/mL). IL-1ß did not affect TNF- production in naive
cells. In the LPS- or IL-1ß-tolerant groups after restimulation with
LPS, a significant decrease in TNF- [353±72pg/mL vs.
866±237pg/mL, respectively (n=3)] was observed.
 View larger version (16K): [in a new window] |
Figure 12. Cell-associated TNF- content. THP-1 cells were rendered LPS (1
µg/mL) or IL-1ß (100 ng/mL) tolerant by pretreatment (pretr.) with
the stimuli or medium alone (naive) for 18 h and subsequently
stimulated with medium alone (basal), LPS (10 µg/mL), or IL-1ß (100
ng/mL) for 24 h. TNF- was assessed in the cell lysates as
described in Materials and Methods. Data represent means ±
SE from three independent experiments. *, P <
0.05 compared with basal (naive) group; #, P < 0.05
compared with LPS stimulation in naive group.
|
 View larger version (21K): [in a new window] |
Figure 13. Effect of pretreatment with different LPS concentrations on
TxB2 production in naive and tolerant cells.
THP-1 cells were rendered LPS tolerant by pretreatment with LPS (1, 10,
or 100 ng/mL or 1000 ng/mL) or medium alone (naive) for 18 h and
then stimulated with LPS (10 µg/mL) for another 18 h. Data
represent means ± SE from three independent
experiments. *, P < 0.05 compared with basal (naive) group;
#, P < 0.05 compared with LPS stimulation in naive group.
|
or IL-1ß. Basal
production of TxB2 was arbitrarily assigned the value of
one (Fig. 14
). LPS stimulation induced a significant increase in
TxB2 production in naive cells (5.6-±1.2-fold;
n=3; P<0.05) compared with basal values.
However, neither TNF- nor IL-1ß stimulated an increase in
TxB2 in naive cells. Pretreatment with TNF- or IL-1ß
induced cross-tolerance to LPS restimulation. Diminished
TxB2 synthesis was observed in TNF--, IL-1ß-, and
LPS-tolerant groups after restimulation with LPS [TxB2
production was reduced 53.5±12.5%, 35.7±17%, and 62.5±5.3%
(n=3), respectively].
 View larger version (21K): [in a new window] |
Figure 14. TxB2 production in LPS-, TNF--, or
IL-1-ß-pretreated cells. TxB2 production was
evaluated in the supernatant of THP-1 cells. Cells were pretreated
(pretr.) with medium alone (naive), LPS (1 µg/mL), TNF- (10
ng/mL), or IL-1ß (100 ng/mL) for 18 h and subsequently
stimulated with LPS (10 µg/mL), TNF- (10 ng/mL), IL-1ß (100
ng/mL), or medium alone (basal) for 18 h.
TxB2 was assessed as described in Materials and
Methods. Data represent means ± SE from three
independent experiments. *, P < 0.05 compared with basal
(naive) group; #, P < 0.05 compared with LPS stimulation in
naive group.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREGULATION OF MAP KINASES...DISCUSSIONREFERENCES |
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, and LPS activate
signaling in naive THP-1 cells as evidenced by ERK and JNK
phosphorylation, IB degradation and DNA p50/p65 NF-B
heterodimer binding. However, unlike LPS, neither TNF- nor IL-1ß
induced IRAK degradation, and these cytokines failed to induce
measurable increases in TNF- and TxB2 production.
Pretreatment of TNF-, IL-1ß, or LPS each induced homologous
tolerance to restimulation with the same agonist. This was demonstrated
by the decrease in ERK and JNK phosphorylation, IB degradation,
and NF-B activation. LPS induced a strong cross-tolerance to TNF-
and IL-1ß signaling events. Because this cross-tolerance was evident
at LPS pretreatment concentrations as low as 10 and 100 ng/mL, it is
not a consequence of the magnitude of the initial LPS stimulus. TNF-
induced some degree of cross-tolerance to LPS stimulation shown by
decreases in ERK phosphorylation and TxB2 production. The
major finding in our study was that IL-1ß tolerance did not produce
cross-tolerance to any of the measured LPS-induced signaling events;
nevertheless, IL-1ß tolerance strongly suppressed LPS-induced
TxB2 and TNF- synthesis.
Previous studies have shown that an ability of LPS and IL-1ß to
induce cross-tolerance to one another was associated with suppression
of activator protein-1 (AP-1) and NF-B transactivation
[18
]. Decreased activation of these transcription
factors could therefore provide a mechanism of suppressed LPS-induced
TNF- and TxB2 production observed in the present study
with IL-1ß-tolerant cells. However, effects of IL-1ß pretreatment
on blocking upstream signal transduction events were not evident in our
study. The data demonstrated that IL-1ß pretreatment does not alter
LPS-induced activation of MAP kinases, IB degradation, or NF-B
translocation. The latter suggests that alternative signaling pathways
may exist which affect gene transactivation. Other studies have
implicated an imprecise relationship between IB degradation and
NF-B DNA binding and transactivation [32
]. Numerous
other signaling pathways including protein kinase C, protein kinase A,
phosphatidylcholine-specific phospholipase, sphingomyelinase, and
tyrosine kinase have been implicated [33
34
35
]. For
example, IL-1ß activation of a NF-B reporter gene in human
alveolar epithelial cells was suppressed by a phospholipase C or
protein kinase C inhibitor, but IL-1ß-induced IB degradation
and NF-B DNA binding were unaffected [36
].
Because our data demonstrated that IL-1ß does not induce TNF-
production, TNF- could not be implicated as an autocrine factor in
inducing cross-tolerance to LPS. It has also been observed that TNF-
is not linked to LPS- or IL-1ß-induced cross-tolerance because
TNF- and LPS do not induce reciprocal tolerance in murine
macrophages [18
]. Also, macrophages from TNFR I/II
knockout mice could be rendered LPS tolerant, and blocking endogenous
TNF- production with TNFR-Fc fusion protein did not alter tolerance
induction . Our results demonstrated that TNF- induces a degree of
cross-tolerance to LPS in THP-1 cells. Thus murine macrophages and
human THP-1 cells may differ in this respect. In THP-1 cells TNF-
tolerance suppressed LPS-induced ERK phosphorylation and induced an
NF-B-binding pattern consisting of increased p50 homodimers similar
to LPS tolerance. TNF- pretreatment also attenuated LPS-induced
TxB2 synthesis. Our previous studies have demonstrated that
TNF- pretreatment in vivo results in cross-tolerance to LPS-induced
lethality in rats and induces a macrophage functional phenotype typical
of LPS tolerance [16
].
LPS tolerance has been shown to suppress murine macrophage expression
of TLR4 and this has been postulated as a mechanism of tolerance
[37
]. However the ability of LPS to induce
cross-tolerance to TNF-- and IL-1ß-induced signaling events, which
occur independently of TLR4, argues that LPS tolerance alters
postreceptor signaling mechanisms. The level at which LPS tolerance may
be affecting the signaling cascade is uncertain. The ability of phorbol
myristate acetate to restore the signaling responses in tolerant
macrophages or monocytes shown by our studies [8
,
11
] and others [38
] suggests proximal
signaling alterations. In a study by Li et al. [39
],
LPS-tolerant THP-1 cells were shown to have impaired signaling at the
level of IRAK. This was evident from decreased LPS-induced IRAK
association with MyD88, decreased IRAK phosphorylation, and decreased
IRAK content. Our studies confirmed their observations that LPS induces
IRAK degradation. IRAK was depleted at 2 h after LPS stimulation
and remained depleted after 18 h, i.e., when the cells were LPS
tolerant. Cellular content of MyD88 was not affected by the LPS
pretreatment (data not shown). However, we demonstrated that neither
TNF- or IL-1ß stimulation nor tolerance affected the IRAK levels.
Thus, it did not appear that IRAK depletion would be a limiting factor
in cytokine-induced cross-tolerance to LPS. IL-1ß has been shown to
induce IRAK degradation in other cell lines [40
]. The
observed lack of IL-1ß pretreatment on IRAK degradation in the
present study is consistent with the observation of Li et.al.
[39
], who demonstrated that IL-1ß had no effect on
IRAK protein levels or IRAK kinase activity in THP-1 cells.
In summary, these data demonstrate that LPS tolerance induces strong
cross-tolerance to TNF-- and IL-1ß-induced cell signaling events.
TNF- tolerance induced partial cross-tolerance to LPS-induced cell
signaling and TxB2 production (Fig. 15
). However, surprisingly, we found that IL-1ß-induced
cross-tolerance to LPS was unique. IL-1ß pretreatment decreased
LPS-stimulated TxB2 and TNF- production (secreted and
cell associated). However, IL-1ß pretreatment did not suppress
LPS-induced ERK and JNK activation, IB degradation, or
NF-B-DNA binding. Therefore, these data demonstrate that IL-1ß is
capable of inducing cross-tolerance to LPS-induced mediator production
without altering LPS-induced activation of MAP kinase signaling
pathways or NF-B activation. It is possible that IL-1ß may affect
more downstream signaling events that impact LPS-induced gene
transactivation and/or posttranscriptional events leading to suppressed
TxB2 and TNF- production. Further studies examining
signaling events and gene expression in LPS tolerance and
cross-tolerance phenomena are merited.
 View larger version (27K): [in a new window] |
Figure 15. Schematic summary of key findings.
|
Received September 11, 2000; revised June 12, 2001; accepted June 18, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSREGULATION OF MAP KINASES...DISCUSSIONREFERENCES |
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