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Tumor Necrosis Factor Induces Lipopolysaccharide Tolerance
in a Human Adenocarcinoma Cell Line Mainly through the
TNF p55 Receptor*
(Received for publication, March 29, 1995, and in revised form, July 9, 1995)
Astrid Lægreid‡§, Liv Thommesen§, Tove Gullstein Jahr‡, Anders Sundan‡, and Terje Espevik‡¶
From the ‡Institute of Cancer Research and Molecular Biology and §UNIGEN, Center for Molecular Biology,
University of Trondheim, N-7005 Trondheim, Norway
This study demonstrates that lipopolysaccharide
(LPS) mediates induction of transcription factor NF
k
B
and activation of the cytomegalovirus (CMV) promoter-
enhancer in the SW480 cell line. These cells do not ex-
press a functional membrane CD14. The LPS response in
SW480 cells was weaker and markedly slower than the
tumor necrosis factor (TNF) response. Pretreatment
with TNF for 72 h inhibited both TNF, tumor necrosis
factor receptor (TNFR) p55, TNFR p75, and LPS-medi-
ated activation of nuclear factor -
k
B (NF
k
B), whereas
pretreatment with LPS only inhibited the LPS response.
TNFR p55 antibody pretreatment resulted in marked
inhibition of the LPS response, while pretreatment with
TNFR p75 antiserum only had a weak inhibitory effect.
Flowcytometric analysis showed that LPS binding as
well as expression of TNFR p55 and TNFR p75 were not
affected by LPS or TNF pretreatment, indicating that
the observed inhibition is not due to reduction of spe-
cific binding sites at the cell surface. The results suggest
that LPS signaling in SW480 cells involves intracellular
components which may be depleted or inactivated via
TNFR p55, indicating that the LPS and TNFR p55 path-
ways overlap. We propose that TNFR p55 can mediate
activation of NF
k
B and cytomegalovirus promoter-en-
hancer in SW480 cells via two distinct mechanisms, one
which is activated only via TNFR p55 and leads to rapid
activation of NF
k
B, and another which is overlapping
with the LPS pathway.
Lipopolysaccharide (LPS),
1
a major membrane component of
Gram-negative bacteria, plays an important role in the patho-
genesis of Gram-negative sepsis leading to septic shock (1).
LPS is a potent stimulator of monocytes and macrophages
which respond by production of tumor necrosis factor (TNF),
interleukin (IL)-1, IL-6, IL-8, eicosanoids (2), and nitric oxide
(3). LPS activates monocytes and macrophages via CD14, a
glycosyl-phosphatidylinositol-anchored surface protein (4).
However, LPS receptors other than CD14 may also contribute
to LPS signaling (5–7).
A wide variety of other cell types are also affected by LPS,
and some of these cells do not express membrane CD14. Thus,
LPS has been reported to stimulate arachidonate metabolism
and surface expression of adhesion molecules in endothelial
cells, and it can induce aggregation of platelets, stimulate
cytokine release from mast cells and fibroblasts, and lead to
generation of chemotactic factors in epithelial cells (8). Al-
though CD14 is not present on the plasma membrane of these
cells, soluble (s)CD14 present in serum is essential for their
stimulation by LPS (9, 10).
Transcription factors activated by LPS include NF
k
B (10, 11)
and NF-IL6 (12, 13). TNF is also known as a potent inducer of
NF
k
B as well as of other transcription factors including AP-1,
NF-IL6, IRF-1, and NF-GMa (14). NF
k
B belongs to the rel
family of transcription factors which form a number of different
hetero- and homodimers participating in the regulation of a
large number of genes involved in the immune response (15).
NF
k
B proteins are constitutively expressed in the cytoplasm,
bound to inhibitor I
k
B, and are released and translocated to the
nucleus upon phosphorylation and degradation of I
k
B (16–18).
Most likely, more than one I
k
B kinase is involved, and different
stimuli lead to phosphorylation and degradation of different
I
k
B species. Thus, while I
k
B-
a
is degraded both by TNF and
LPS, I
k
B-
b
is only degraded in response to LPS and IL-1, but
not in response to TNF (19).
TNF can induce NF
k
B via two different receptors, TNFR p55
and TNFR p75 (20). In most cells, TNFR p55 is responsible for
activation of NF
k
B (21, 22), and recent evidence indicates that
TNFR p55 signaling involves ceramide (23, 24). In some cell
types, NF
k
B may also be activated by TNFR p75 (20), but
TNFR p75 signaling mechanisms are still poorly understood.
Association of serine/threonine kinases with cytosolic domains
of both TNFR p75 (25) and TNFR p55 (26, 27) have been
reported, and recently, two cytosolic proteins which specifically
associate with TNFR p75 intracellular domains were identified
and cloned (28). However, no intracellular pathways activated
by any of these proteins have yet been identified, and it is not
known whether they are involved in TNF-mediated activation
of NF
k
B.
LPS-mediated activation of NF
k
B in monocytes is dependent
on intracellular protein tyrosine kinase activity (29). Similarly,
TNF-mediated activation of NF
k
B in lysates of the monocyte-
derived cell line U937 was inhibited by a tyrosine kinase in-
hibitor (30). Also, both LPS- and TNF-induced activation of
NF
k
B is inhibited by the antioxidant pyrrolidine dithiocarba-
mate (31, 32) indicating that reactive oxygen intermediates
play a role in both LPS- and TNF-mediated NF
k
B activation.
So far, LPS signal transduction has been most extensively
studied in cells expressing a functional membrane CD14. How-
ever, LPS effects on CD14 negative cell types may also contrib-
* This work was supported by The Norwegian Cancer Society and by
The Research Council of Norway and Pronova Biopolymer. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶To whom correspondence should be addressed: Institute of Cancer
Research and Molecular Biology, University of Trondheim, N-7005
Trondheim, Norway. Tel.: 47-73-59-86-68; Fax: 47-73-59-88-01.
1
The abbreviations used are: LPS, lipopolysaccharide;
b
-gal,
b
-galac-
tosidase; CMV, cytomegalovirus; mAb, monoclonal antibody; PC-PLC,
phosphatidyl-specific phospholipase C; PMA, phorbol 12-myristate 13-
acetate; p75 AS, TNFR p75 antiserum; TNF, tumor necrosis factor;
TNFR tumor necrosis factor receptor; IL, interleukin; FITC, fluorescein
isothiocyanate; LBP, LPS-binding protein.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 43, Issue of October 27, pp. 25418–25425, 1995
© 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
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ute to LPS-mediated pathology. In the present study, we show
that LPS activates the transcription factor NF
k
B and the cy-
tomegalovirus (CMV) promoter-enhancer in SW480 human ad-
enocarcinoma cells which do not have a functional membrane
CD14. Since both TNFR p55 and TNFR p75 independently
activate SW480 cells (20), we considered it important to com-
pare the LPS signaling mechanism to the signal transduction
pathways activated by the TNF receptors. The results demon-
strate that TNF induces LPS tolerance in SW480 cells and that
TNFR p55 and LPS may activate overlapping pathways.
MATERIALS AND METHODS
Cell Cultivation and Stimulation—Human colon adenocarcinoma
cells, SW480/
b
-gal (generously provided by Dr. Gerald Ranges, Miles
Inc., West Haven, CT), contain a
b
-galactosidase (
b
-gal) gene under the
control of the CMV immediate early promoter-enhancer (33). SW480/
b
-gal were grown in RPMI 1640 (Life Technologies, Inc. Laboratories,
Paisley, Scotland), supplemented with 2 mML-glutamine, 10% heat-
inactivated fetal calf serum (HyClone, Logan, UT), and 40
m
g/ml gara-
mycin (fetal calf serum medium). Stimulation with LPS was routinely
carried out in RPMI 1640 medium supplemented with glutamine, 20%
human A
1
serum (The Blood Bank, University Hospital of Trondheim,
Trondheim, Norway), and garamycin (A
1
medium). Experiments con-
ducted at serum-free conditions were performed in AIM medium (Life
Technologies, Inc.).
Reagents—LPS from smooth Salmonella minnesota 6261 and from
rough S. minnesota Re595 (Sigma) as well as S. minnesota lipid A di-
and monophosphate (Sigma) were solubilized in 0.9% NaCl at stock
solutions of 2 mg/ml (LPS) or 1 mg/ml (lipid A). Recombinant (r)CD14
and (r)LBP were generously provided by Dr. M. Lichenstein and Dr. M.
Zukowski, AMGEN (Thousand Oaks, CA) and were prepared as de-
scribed (34). Human recombinant TNF, with specific activity 7.6 310
7
units/mg protein, was generously supplied by Dr. Refaat Shalaby,
Genentech Inc. (South San Francisco, CA). Anti-CD14 monoclonal an-
tibody (mAb) 3C10 was obtained from a subclone of the hybridoma
ATCC TIB 228 (American Type Culture Collection).
TNFR p75 antiserum (p75 AS) was generated by multiple injections
of a rabbit with recombinant soluble TNFR p75 (20). The mAb htr-9
against TNFR p55 (35) was generously provided by Dr. M. Brockhaus,
Hoffmann La-Roche Ltd. (Basel, Switzerland). Biotinylation of htr-9
and mAb utr-4 against TNFR p75 (35) was performed as described (36).
The mAb 6H8 directed against a widely distributed 180-kDa membrane
protein
2
was used as a control antibody. All mAbs were purified on a
Sepharose goat anti-mouse IgG column (Zymed Laboratories Inc.,
South San Francisco, CA). Benzamidine (Sigma) was dissolved in 50%
ethanol at 0.5 Mand phenylmethylsulfonyl fluoride (Sigma) in isopropyl
alcohol at 0.1 M.
b
-Galactosidase Assay—The
b
-galactosidase assay was performed
essentially as described previously (20). Substrate conversion was
measured as optical density (OD) at 570 nm. For pretreatment studies,
cells were seeded out in A
1
medium containing pretreatment reagents.
After 72 h, the plates were washed three times in Hanks’ buffered salt
solution, and test reagents were added in A
1
medium.
Quantitative Band Shift Assays—Preparation of nuclear extracts
and band shift analysis was performed essentially as described (20).
Briefly, equal amounts of nuclear protein from each sample were incu-
bated with 1
m
g of poly(dI-dC) (Pharmacia Fine Chemicals, Uppsala,
Sweden), in binding buffer (20 mMHEPES, pH 7.9, 50 mMKCl, 1 mM
EDTA, 1 mMdithiothreitol, 0.25 mg/ml bovine serum albumin, 2%
Ficoll) (20
m
l final volume) for 10 min at room temperature. Then, 17
fmol (1–5 310
4
counts/min) of end-labeled NF
k
B-specific double-
stranded oligonucleotide probe 59-AGTTGAGGGGACTTTCCCAGG-39
(Promega Corp., Madison, WI) was added, and the mixture was incu-
bated for another 15–20 min. The samples were applied on native
polyacrylamide gels (7% acrylamide, 0.25 3Tris borate-EDTA, 2.5%
glycerol) and run at 100 V for 1 h and then at 130 V for 2–3 h, after
which the gels were dried and exposed to x-ray film (X-Omat AR,
Kodak, Rochester, NY) for 2–16 h. Specific band shifts were quantitated
with a PhosphoImager (Molecular Dynamics, Sunnyvale, CA) by meas-
uring the radioactivity within a rectangle enclosing the band shift in gel
scans after exposures of 18–24 h. Radioactivity counts, which are a
2
B. Naume, A. Sundan, and T. Espevik, unpublished results.
FIG.1. Induction of
b
-galactosidase activity in SW480/
b
-gal
cells by LPS from S.minnesota,E.coli or Pseudomonas,orbyS.
minnesota lipid A.
b
-Galactosidase activity was measured after stim-
ulation of the cells for4hinA
1
medium. Results (mean values of
duplicates) of a representative experiment are given.
FIG.2.Serum dependence and effect of CD14 mAb, rCD14, and rLBP on Re595 LPS-induced
b
-galactosidase activity. SW480/
b
-gal
cells were stimulated for 4 h with Re595 LPS in medium with increasing amounts of human serum (A), in A
1
medium (20% human serum) with
or without neutralizing CD14 mAb 3C10 or control mAb 6H8 (B), or in serum-free AIM medium with or without the addition of recombinant CD14,
recombinant LBP or 20% human serum (C). Results (mean values of duplicates) of a representative experiment are given.
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function of both gel exposure times and of the specific activity of the
probe, were termed “PhosphoImager units” and were used to compare
the relative amounts of radioactivity in band shifts within one gel. In
order to control that a similar amount of nuclear proteins was applied
for each sample, we (i) performed band shift assays with
32
P-labeled
OCT oligonucleotide (Promega) (59-TGTCGAATGCAAATCACTAGAA-
39) which binds to constitutively expressed “octamer” proteins (37), or
(ii) measured the intensity of nonspecific bands in the NF
k
B band shift.
All samples were analyzed in at least two band shift assays.
Flow Cytometric Analysis of TNFR mAb and FITC-LPS Binding—
SW480/
b
-gal cells were pretreated with 10 ng/ml TNF or with 0.1
m
g/ml
Re595 LPS in A
1
medium for 72 h before the cells were labeled with 10
m
g/ml of biotinylated (B)htr-9 or Butr-4 and 0.2
m
g/ml streptavidin-
phycoerythrin (Becton Dickinson, Mountain View, CA) as described
previously (38). Binding of FITC-LPS was estimated by adding 10
m
g/ml
FITC-labeled LPS from S. minnesota 6261 in PBS with 2.5% A
1
serum
(PBS-A
1
buffer) to 1 310
6
SW480/
b
-gal cells for 40 min at 0–4 °C.
Background fluorescence was estimated by adding 6.2
m
g/ml of FITC-
goat anti-murine Ig (Becton Dikinson). After washing three times
in PBS-A
1
buffer, cells were analyzed in a FACScan flow cytometer
(Becton Dickinson).
RESULTS
LPS Activates the CMV Promoter-enhancer in the Colon Car-
cinoma Cell Line SW480—Transcriptional activation of the
CMV promoter-enhancer was measured in a reporter gene
assay in SW480 cells stably transfected with a plasmid con-
taining
b
-gal under control of the CMV promoter-enhancer
(33). Treatment of SW480/
b
-gal cells with LPS resulted in
strong induction of
b
-gal activity (Fig. 1) which shows that LPS
can activate intracellular signals leading to activation of the
CMV promoter-enhancer in SW480 cells. S.minnesota LPS
was found to be several orders of magnitude more potent than
LPS from Escherichia coli or from Pseudomonas. S. minnesota
mutant Re595 LPS gave rise to a higher maximal response
than wild type 6261 LPS, while the lipid A part of S. minnesota
LPS showed very little activity on its own (Fig. 1).
The LPS activity was strongly influenced by human serum
which caused a dose-dependent enhancement of LPS mediated
b
-gal activity (Fig. 2A). The presence of neutralizing anti-CD14
mAb 3C10 completely inhibited LPS-mediated induction of
b
-gal activity (Fig. 2B), and we found that recombinant
(r)CD14 could enhance LPS activity in serum-free medium
(Fig. 2C). Recombinant LPS-binding protein (LBP) had little or
no enhancing effect on the LPS response (Fig. 2C), and LBP did
not further potentiate the rCD14-mediated enhancement of the
LPS response (Fig. 2C). Taken together, the data indicate that
SW480 cells do not express a functional membrane CD14 and
that soluble (s)CD14 in human serum is necessary for the LPS
response. The LPS effect was not mediated by TNF or LT-
a
as
neutralizing antibodies against TNF or LT-
a
did not inhibit the
LPS response (data not shown).
LPS Induces Activation of Transcription Factor NF
k
Bata
Significantly Slower Rate than TNF—Nuclear extracts from
SW480 cells stimulated with LPS or TNF were analyzed for
trancription factor NF
k
B. LPS was found to induce activation
of NF
k
B in a dose-dependent manner, and S. minnesota Re595
mutant LPS was more potent than wild type (6261) LPS (Fig.
3). The response maximum of LPS was markedly lower than
that of TNF.
The finding that LPS and TNF both induce activation of
FIG.3.Activation of NF
k
B in SW480/
b
-gal cells by S. minnesota
6261 LPS, Re595 LPS, and TNF. A,NF
k
B band shift analysis of
nuclear extracts from cells stimulated for 2 h with increasing amounts
of TNF (lanes 1–6), Re595 LPS (lanes 7–12), or 6261 LPS (lanes 13–18),
in A
1
medium. The two bands marked with arrowheads represent
nuclear proteins binding specifically to the NF
k
B consensus sequence,
as identified previously (20). The faster migrating complex () is only
weakly up-regulated and mainly contains NF
k
B p50 as judged by
supershift analysis.
3
The slower migrating complex () contains both
NF
k
B p50 and p65
3
and is strongly up-regulated. B, OCT band shift
analysis of the same extracts as in A.C, quantitation of relative radio-
activity in the slower migrating, strongly up-regulated p50/p65 NF
k
B
complex () from the band shift analysis shown in A, by PhosphoImager
measurements (mean 6S.D. of triplicate measurements of the same
band shifts). Similar results were obtained by analysis of two other
series of nuclear extracts from cells stimulated as above.
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transcription factor NF
k
B and activate the CMV promoter-
enhancer in SW480 cells, raises the question whether these
agents mediate their effects through similar intracellular
mechanisms. In order to compare the LPS and TNF responses,
we first analyzed the kinetics of NF
k
B activation for each of the
stimuli. It was found that LPS activated NF
k
B at a signifi-
cantly slower rate than TNF (Fig. 4, Aand B). Activation of
NF
k
B by TNF was clearly detectable at 10 min and reached a
plateau level after approximately 45 min. However, LPS-in-
duced NF
k
B activation was only detected after 60 min and
further increased during incubations of up to 120 min (Fig. 4, A
and B). Stimulation with LPS for more than 2 h did not signif-
icantly increase the amounts of activated NF
k
B (data not
shown). In the reporter gene assay, LPS required at least6hto
yield maximal response, while the TNF response reached pla-
teau levels after4hofincubation (Fig. 4C), indicating that LPS
also activates the CMV promoter-enhancer at a slower rate
than TNF. These results suggest that LPS activates NF
k
B and
CMV promoter-enhancer via mechanisms that are different
from the TNF-activated mechanisms.
Pretreatment with TNF Inhibits LPS-induced Activation of
NF
k
B but LPS Pretreatment Does Not Inhibit TNFR p55 or
TNFR p75 Responses—LPS- and TNF-induced NF
k
B activa-
tion was further compared in a series of experiments where
SW480 cells were pretreated for 72 h with either LPS, TNF, or
agonistic antibodies against TNFR p55 or TNFR p75, followed
by stimulation with LPS, TNF, or agonistic TNFR antibodies.
Pretreatment of the cells for 72 h with TNF only marginally
influenced the expression of TNFR p55 and TNFR p75 recep-
tors (Fig. 5A). Furthermore, pretreatment of the cells with TNF
or LPS did not affect the binding of FITC-LPS from S. minne-
sota (Fig. 5B).
Band shift analysis of nuclear extracts from pretreated cells
stimulated with LPS, TNF, or with agonistic anti-TNFR anti-
bodies are shown in Fig. 6. The data demonstrate that the LPS
response is inhibited by pretreatment with either LPS or with
TNF or agonistic TNFR antibodies (Fig. 6, Aand B), while the
TNF response is only inhibited by pretreatment with TNF or
TNFR antibodies and not by LPS pretreatment (Fig. 6, Cand
D). Activation of NF
k
B by TNFR p55 mAb htr-9 was inhibited
by pretreatment with htr-9 or TNF (Fig. 6, Eand F), while the
TNFR p75-mediated response was mainly inhibited by p75 AS
and TNF pretreatment (Fig. 6, Gand H). Taken together, the
results suggest that pretreatment with a given stimulus leads
to depletion or reduction of active intracellular components
involved in the signal transduction pathway induced by that
stimulant. In addition, pretreatment with agents like htr-9 or
TNF may also reduce the level of active components involved in
signal transduction by other agents like LPS.
In all experiments performed, inhibition of the LPS response
by pretreatment with TNFR p55 mAb htr-9 was of a similar
magnitude as inhibition by LPS pretreatment (Fig. 6, Aand B),
indicating that activation of NF
k
B by LPS is dependent on
cellular components which are reduced or inactivated by long
term stimulation of TNFR p55. Pretreatment with TNFR p75
antiserum led to a weak but consistent inhibition of the LPS
response (Fig. 6, Aand B) and thus, components activated via
TNFR p75 may also be involved in LPS signal transduction.
However, neither TNFR p55- nor TNFR p75-mediated activa-
tion of NF
k
B was inhibited by pretreatment with LPS (Fig. 6,
E-H), suggesting that long term stimulation with LPS does not
lead to a reduction or inactivation of components involved in
either of these pathways. In contrast to the lack of inhibition of
the TNFR p75 response by LPS, pretreatment with TNFR p55
mAb htr-9 caused a low but reproducible inhibition of the
TNFR p75 response. This inhibition, however, was markedly
FIG.4.Kinetics of Re595 LPS- and TNF-mediated activation of NF
k
B and CMV promoter-enhancer in SW480/
b
-gal cells. A, band
shift analysis of nuclear extracts from cells treated for the indicated time points with either Re595 LPS (1
m
g/ml) or with TNF (10 ng/ml) in A
1
medium. The two specific NF
k
B complexes are indicated as in Fig. 3. B, PhosphoImager quantitation of radioactivity in the slower migrating
p50/p65 NF
k
B complex (mean 6S.D. of triplicate measurements of the same band shifts). Similar results were obtained by analysis of two other
series of nuclear extracts from cells stimulated as above. C, induction of
b
-galactosidase activity in cells treated for the indicated time points with
either Re595 LPS (0.1
m
g/ml), TNF (10 ng/ml), or A
1
medium. Results (mean values of duplicates) of a representative experiment are given.
FIG.5.Flowcytometric analysis of TNFR mAb and FITC-LPS
binding to pretreated SW480/
b
-gal cells. Cells were pretreated for
72 h with TNF (1 ng/ml) or LPS (0, 1
m
g/ml) followed by labeling of the
cells with p55/p75 mAb (A) or FITC-LPS (B) as described under
“Materials and Methods.”
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lower than the inhibition of the TNFR p75 response caused by
p75 AS pretreatment (Fig. 6, Gand H). The TNFR p55-medi-
ated response was not inhibited by pretreatment with p75 AS
in any of the experiments. Thus, it seems that TNFR p55-
induced activation of NF
k
B does not depend on components
activated via TNFR p75.
Pretreatment with TNFR p55 Agonistic Antibodies Can Also
Suppress LPS-induced Transcriptional Activation of the CMV
Promoter-enhancer—LPS- and TNFR-mediated activation of
the CMV promoter-enhancer was compared by subjecting the
cells to similar protocols of pretreatment and stimulation as
described above. It was found that LPS-induced
b
-gal activity
was markedly reduced after pretreatment with LPS or with the
TNFR p55 mAb htr-9, while pretreatment with p75 AS had less
inhibitory effect (Fig. 7A). The TNFR p55-mediated activity
was almost completely abolished after pretreatment with
TNFR p55 mAb htr-9 and very weakly inhibited by pretreat-
ment with p75 AS, while pretreatment with LPS showed no
inhibitory effect (Fig. 7B). Thus, pretreatment of the cells af-
fected the subsequent activation of both the CMV promoter-
enhancer and NF
k
B in a similar manner.
DISCUSSION
The present paper demonstrates that LPS can induce acti-
vation of transcription factor NF
k
B, as well as activation of the
CMV promoter-enhancer in the human adenocarcinoma cells
SW480. These cells do not express a functional membrane
CD14 because addition of LBP did not enhance the LPS effect
under serum free conditions. Such lack of enhancement is a
typical phenomenon in cells which do not express a functional
membrane CD14 (39, 40). Other CD14 negative cells where
LPS has been found to mediate activation of NF
k
B include the
murine pre-B-cell line 70Z/3 (10) and endothelial cells (11). The
LPS response in SW480 cells was strongly dependent on hu-
FIG.6.Activation of NF
k
B in pretreated SW480/
b
-gal cells by Re595 LPS, TNF, or agonistic TNFR antibodies. Cells were pretreated
of for 72 h with either A
1
medium alone, Re595 LPS (0.1
m
g/ml), TNF (1 ng/ml), TNFR p55 mAb htr-9 (10
m
g/ml) (35), or with TNFR p75 antiserum
p75 AS (dil. 1/100) (20), and band shift analysis performed on nuclear extracts from cells stimulated for 2 h with different doses of Re595 LPS (A),
TNF (C), htr-9 (E), or with p75 AS (G), followed by PhosphoImager quantitation of relative radioactivity in the slower migrating, p50/p65
containing complex B,D,F, and H(mean 6S.D. of triplicate measurements of the same band shifts). Similar results were obtained by analysis
of three other series of nuclear extracts from cells pretreated and stimulated as above.
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man serum and could be completely inhibited by neutralizing
antibodies against CD14. Recombinant soluble (s)CD14 could
only partly compensate for the human serum enhancing effect
since the activity of LPS in the presence of rCD14 was mark-
edly lower than LPS activity in the presence of 20% human
serum. Thus, sCD14 is necessary but not sufficient for LPS
activity, and other serum factors in addition to sCD14 are
necessary for maximal LPS response in SW480 cells.
LPS stimulates activation of NF
k
B at a markedly slower rate
than TNF, indicating that the LPS and TNF signal transduc-
tion pathways are not identical. As shown earlier, the kinetics
of NF
k
B activation by agonistic antibodies against the TNFR
p55 is identical to the TNF kinetics with maximal levels
reached after ;45 min, while stimulation of TNFR p75 results
in maximum activation after ;60 min (20). Thus, in order to
reach maximum NF
k
B activation, stimulation with LPS has to
be continued for a significantly longer period of time than
stimulation of any of the TNF receptors, indicating that the
LPS signaling mechanism differs from the mechanisms em-
ployed by the two TNF receptors. Supershift analysis showed
that stimulation of SW480 cells with LPS or with agonistic
TNFR p55 or p75 antibodies resulted in activation of an iden-
tical pattern of NF
k
B hetero- and homodimers.
3
Thus, SW480
is a cell system where the different pathways employed by LPS
and the two TNF receptors can be compared, and where the
question can be asked as to whether these pathways are inde-
pendent or overlapping.
Comparison of LPS and TNF signal transduction pathways
was performed by analyzing activation of NF
k
B and the CMV
promoter-enhancer in cells pretreated with LPS, TNF, or with
agonistic antibodies against TNFRs. We found that pretreat-
ment of the cells did not result in down-regulation of TNF
receptors or reduction in the binding of LPS. Thus, it is likely
that the observed inhibition of activation of NF
k
B and CMV
promoter-enhancer is due to intracellular effects of the pre-
treatment analogous to depletion of protein kinase C by long
term treatment with the phorbol ester PMA. Such treatment
renders cells unresponsive to subsequent activation of NF
k
Bby
PMA, while the TNF response remains unaffected, indicating
3
A. Lægreid and L. Thommesen, unpublished observations.
FIG.6—continued
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that TNF does not depend on PMA-responsive protein kinase C
for activation of NF
k
B (41, 42).
The results from the pretreatment experiments are in agree-
ment with our previous results which indicated that TNFR p75
mediates NF
k
B activation through a different signal transduc-
tion mechanism than TNFR p55 (20). Thus, the TNFR p75-
mediated response is maximally inhibited by pretreatment
with p75 AS while pretreatment with TNFR p55 mAb htr-9 led
to a markedly lower reduction of the TNFR p75 response.
Furthermore, lack of inhibition of the TNFR p75 response by
LPS pretreatment suggests that the TNFR p75-activated sig-
naling mechanism is independent of components activated by
LPS. Thus, the TNFR p75 pathway leading to NF
k
B activation
is different from the LPS, as well as from the TNFR p55
pathway, although it appears to include intracellular compo-
nents which are depleted or inactivated by long term stimula-
tion of TNFR p55.
TNFR p55, which mediates rapid NF
k
B activation, appar-
ently employs a signaling mechanism which is independent of
intracellular components activated by LPS or TNFR p75,
since pretreatment with LPS or TNFR p75 AS did not inhibit
the TNFR p55 response. On the other hand, the LPS response
seems to be mediated by a pathway which is partly overlap-
ping with the TNFR p55 pathway, since pretreatment with
TNFR p55 mAb htr-9 inhibited the LPS response to a similar
extent as the LPS pretreatment. The observation that TNFR
p55 and LPS signaling pathways may be partly overlapping
suggests that TNFR p55 employs more than one pathway
leading to activation of NF
k
B in SW480 cells. Thus, our
results suggest that TNFR p55 may activate one pathway
which mediates rapid activation of NF
k
B and is independent
of intracellular components activated by LPS or p75 AS, and
another pathway which overlaps with the LPS signal trans-
duction pathway.
The stage at which the LPS and TNFR p55 signaling path-
ways overlap may involve ceramide, a lipid messenger which
participates in the activation of NF
k
B in several cell lines
including Jurkat (23), HL-60 (43), and SW480 (42). TNFR
p55-mediated activation of NF
k
B in Jurkat cells as well as
70Z/3 cells has been found to proceed by ceramide generated by
an acidic sphingomyelinase (23, 44), while TNFR p55-mediated
activation of NF
k
B in HL-60 cells is reported to involve a
97-kDa ceramide-activated protein kinase which is activated
via ceramide generated by a neutral sphingomyelinase (43, 45,
46). Recently, LPS was found to stimulate ceramide-activated
protein kinase in HL-60 cells directly, in the absence of detect-
able sphingomyelinase activity (47). A possible reason for this
LPS activity may be structural similarities between LPS and
ceramide (47). Thus, ceramide-activated protein kinase may be
an intracellular component putatively involved in both LPS-
and TNFR p55-mediated NF
k
B activation in SW480 cells.
Release of LPS during Gram-negative infections may induce
high levels of circulating TNF which can lead to shock and
death (48). Our finding that TNF pretreatment inhibits LPS-
induced NF
k
B activation may have important clinical implica-
tions as release of low TNF levels during Gram-negative infec-
tions could render cells resistant to subsequent LPS
stimulation. This is supported by in vivo data showing that
pretreatment of mice with TNF or IL-1 induces partial toler-
ance to LPS (49). Thus, release of low TNF levels during Gram-
negative infections may have an important function in limiting
harmful effects of LPS in vivo.
Acknowledgments—We thank Siv Moen, Wenche Rikardsen, Liv
Ryan, and Mari Sørensen for excellent technical assistance.
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Astrid Lægreid, Liv Thommesen, Tove Gullstein Jahr, Anders Sundan and Terje Espevik
Adenocarcinoma Cell Line Mainly through the TNF p55 Receptor
Tumor Necrosis Factor Induces Lipopolysaccharide Tolerance in a Human
doi: 10.1074/jbc.270.43.25418
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