(Received for publication, March 29, 1995; and in revised form, July 9, 1995)
From the
This study demonstrates that lipopolysaccharide (LPS) mediates
induction of transcription factor NFB and activation of the
cytomegalovirus (CMV) promoter-enhancer in the SW480 cell line. These
cells do not express 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-mediated activation of nuclear factor -
B (NF
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 specific 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 pathways overlap. We propose that
TNFR p55 can mediate activation of NF
B and cytomegalovirus
promoter-enhancer in SW480 cells via two distinct mechanisms, one which
is activated only via TNFR p55 and leads to rapid activation of
NF
B, and another which is overlapping with the LPS pathway.
Lipopolysaccharide (LPS), ()a major membrane
component of Gram-negative bacteria, plays an important role in the
pathogenesis 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, 6, 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) . Although 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 NFB (10, 11) and
NF-IL6(12, 13) . TNF is also known as a potent inducer
of NF
B as well as of other transcription factors including AP-1,
NF-IL6, IRF-1, and NF-GMa(14) . NF
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
B
proteins are constitutively expressed in the cytoplasm, bound to
inhibitor I
B, and are released and translocated to the nucleus
upon phosphorylation and degradation of
I
B(16, 17, 18) . Most likely, more than
one I
B kinase is involved, and different stimuli lead to
phosphorylation and degradation of different I
B species. Thus,
while I
B-
is degraded both by TNF and LPS, I
B-
is
only degraded in response to LPS and IL-1, but not in response to
TNF(19) .
TNF can induce NFB via two different
receptors, TNFR p55 and TNFR p75(20) . In most cells, TNFR p55
is responsible for activation of NF
B(21, 22) ,
and recent evidence indicates that TNFR p55 signaling involves
ceramide(23, 24) . In some cell types, NF
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
B.
LPS-mediated activation of
NFB in monocytes is dependent on intracellular protein tyrosine
kinase activity(29) . Similarly, TNF-mediated activation of
NF
B in lysates of the monocyte-derived cell line U937 was
inhibited by a tyrosine kinase inhibitor(30) . Also, both LPS-
and TNF-induced activation of NF
B is inhibited by the antioxidant
pyrrolidine dithiocarbamate (31, 32) indicating that
reactive oxygen intermediates play a role in both LPS- and TNF-mediated
NF
B activation.
So far, LPS signal transduction has been most
extensively studied in cells expressing a functional membrane CD14.
However, LPS effects on CD14 negative cell types may also contribute to
LPS-mediated pathology. In the present study, we show that LPS
activates the transcription factor NFB and the cytomegalovirus
(CMV) promoter-enhancer in SW480 human adenocarcinoma 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 compare the LPS signaling mechanism to the signal
transduction pathways activated by the TNF receptors. The results
demonstrate that TNF induces LPS tolerance in SW480 cells and that TNFR
p55 and LPS may activate overlapping pathways.
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 ()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 M and
phenylmethylsulfonyl fluoride (Sigma) in isopropyl alcohol at 0.1 M.
Figure 1:
Induction of
-galactosidase activity in SW480/
-gal cells by LPS from S. minnesota, E. coli or Pseudomonas, or by S. minnesota lipid A.
-Galactosidase activity was measured after stimulation of the
cells for 4 h in A
medium. Results (mean values of
duplicates) of a representative experiment are
given.
The LPS activity was strongly influenced by human
serum which caused a dose-dependent enhancement of LPS mediated
-gal activity (Fig. 2A). The presence of
neutralizing anti-CD14 mAb 3C10 completely inhibited LPS-mediated
induction of
-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-
as neutralizing antibodies
against TNF or LT-
did not inhibit the LPS response (data not
shown).
Figure 2:
Serum
dependence and effect of CD14 mAb, rCD14, and rLBP on Re595 LPS-induced
-galactosidase activity. SW480/
-gal cells were stimulated for
4 h with Re595 LPS in medium with increasing amounts of human serum (A), in A
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.
Figure 3:
Activation of NFB in SW480/
-gal
cells by S. minnesota 6261 LPS, Re595 LPS, and TNF. A, NF
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
medium. The two
bands marked with arrowheads represent nuclear proteins
binding specifically to the NF
B consensus sequence, as identified
previously(20) . The faster migrating complex (
) is only
weakly up-regulated and mainly contains NF
B p50 as judged by
supershift analysis.
The slower migrating complex (
)
contains both NF
B p50 and p65
and is strongly
up-regulated. B, OCT band shift analysis of the same extracts
as in A. C, quantitation of relative radioactivity in
the slower migrating, strongly up-regulated p50/p65 NF
B complex
(
) from the band shift analysis shown in A, by
PhosphoImager measurements (mean ± S.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.
The finding that
LPS and TNF both induce activation of transcription factor NFB 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
B activation for
each of the stimuli. It was found that LPS activated NF
B at a
significantly slower rate than TNF (Fig. 4, A and B). Activation of NF
B by TNF was clearly detectable at 10
min and reached a plateau level after approximately 45 min. However,
LPS-induced NF
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 significantly increase the amounts of activated NF
B (data not
shown). In the reporter gene assay, LPS required at least 6 h to yield
maximal response, while the TNF response reached plateau levels after 4
h of incubation (Fig. 4C), indicating that LPS also
activates the CMV promoter-enhancer at a slower rate than TNF. These
results suggest that LPS activates NF
B and CMV promoter-enhancer
via mechanisms that are different from the TNF-activated mechanisms.
Figure 4:
Kinetics of Re595 LPS- and TNF-mediated
activation of NFB and CMV promoter-enhancer in SW480/
-gal
cells. A, band shift analysis of nuclear extracts from cells
treated for the indicated time points with either Re595 LPS (1
µg/ml) or with TNF (10 ng/ml) in A
medium. The two
specific NF
B complexes are indicated as in Fig. 3. B, PhosphoImager quantitation of radioactivity in the slower
migrating p50/p65 NF
B complex (mean ± S.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
-galactosidase activity in
cells treated for the indicated time points with either Re595 LPS (0.1
µg/ml), TNF (10 ng/ml), or A
medium. Results (mean
values of duplicates) of a representative experiment are
given.
Figure 5:
Flowcytometric analysis of TNFR mAb and
FITC-LPS binding to pretreated SW480/-gal cells. Cells were
pretreated for 72 h with TNF (1 ng/ml) or LPS (0, 1 µg/ml) followed
by labeling of the cells with p55/p75 mAb (A) or FITC-LPS (B) as described under ``Materials and
Methods.''
Band shift analysis of nuclear
extracts from pretreated cells stimulated with LPS, TNF, or with
agonistic anti-TNFR antibodies 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, A and B), while the TNF response is only inhibited by
pretreatment with TNF or TNFR antibodies and not by LPS pretreatment (Fig. 6, C and D). Activation of NFB by
TNFR p55 mAb htr-9 was inhibited by pretreatment with htr-9 or TNF (Fig. 6, E and F), while the TNFR p75-mediated
response was mainly inhibited by p75 AS and TNF pretreatment (Fig. 6, G and 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.
Figure 6:
Activation of NFB in pretreated
SW480/
-gal cells by Re595 LPS, TNF, or agonistic TNFR antibodies.
Cells were pretreated of for 72 h with either A
medium
alone, Re595 LPS (0.1 µg/ml), TNF (1 ng/ml), TNFR p55 mAb htr-9 (10
µg/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 ± S.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.
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, A and B), indicating that activation of NFB 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, A and B) and thus, components activated via TNFR p75
may also be involved in LPS signal transduction. However, neither TNFR
p55- nor TNFR p75-mediated activation of NF
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 lower than the inhibition of the TNFR p75
response caused by p75 AS pretreatment (Fig. 6, G and H). The TNFR p55-mediated response was not inhibited by
pretreatment with p75 AS in any of the experiments. Thus, it seems that
TNFR p55-induced activation of NF
B does not depend on components
activated via TNFR p75.
Figure 7:
Induction of -galactosidase activity
by Re595 LPS or by agonistic TNFR p55 mAb htr-9 in pretreated
SW480/
-gal cells. Cells were pretreated for 72 h with either
A
medium alone, Re595 LPS (0.1 µg/ml), htr-9 (1
µg/ml), or with TNFR p75 antiserum p75 AS (dilution 1/200) followed
by stimulation for 4 h with Re595 LPS (A) or htr-9 (B). Results (mean values of duplicates) of a representative
experiment are given.
The present paper demonstrates that LPS can induce activation
of transcription factor NFB, 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
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 human 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 markedly 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 NFB at a markedly slower
rate than TNF, indicating that the LPS and TNF signal transduction
pathways are not identical. As shown earlier, the kinetics of NF
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
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 employed
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 identical pattern of NF
B hetero- and
homodimers. (
)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 independent or overlapping.
Comparison of LPS and TNF
signal transduction pathways was performed by analyzing activation of
NFB and the CMV promoter-enhancer in cells pretreated with LPS,
TNF, or with agonistic antibodies against TNFRs. We found that
pretreatment 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
B and CMV
promoter-enhancer is due to intracellular effects of the pretreatment
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
B by PMA, while the TNF response
remains unaffected, indicating that TNF does not depend on
PMA-responsive protein kinase C for activation of
NF
B(41, 42) .
The results from the
pretreatment experiments are in agreement with our previous results
which indicated that TNFR p75 mediates NFB activation through a
different signal transduction 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 signaling mechanism is independent
of components activated by LPS. Thus, the TNFR p75 pathway leading to
NF
B activation is different from the LPS, as well as from the TNFR
p55 pathway, although it appears to include intracellular components
which are depleted or inactivated by long term stimulation of TNFR p55.
TNFR p55, which mediates rapid NFB activation, apparently
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
overlapping 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
B in SW480 cells. Thus, our
results suggest that TNFR p55 may activate one pathway which mediates
rapid activation of NF
B and is independent of intracellular
components activated by LPS or p75 AS, and another pathway which
overlaps with the LPS signal transduction pathway.
The stage at
which the LPS and TNFR p55 signaling pathways overlap may involve
ceramide, a lipid messenger which participates in the activation of
NFB in several cell lines including Jurkat(23) , HL-60 (43) , and SW480(42) . TNFR p55-mediated activation of
NF
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
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 detectable 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
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 NFB activation may have important clinical
implications as release of low TNF levels during Gram-negative
infections 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 tolerance 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.