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INTRODUCTION |
Lipopolysaccharide
(LPS)1 is the major
constituent of the outer membrane of Gram-negative bacteria that plays
a beneficial role in the course of bacterial infection due to its
ability to stimulate the host immune response (1). However, in excess, LPS is harmful because it causes increased secretion of proinflammatory cytokines (e.g. TNF, IL-1
, and IL-6) that contribute to
septic shock (2). Several LPS-binding proteins have been identified, yet the true LPS signaling receptor(s) and the mechanism by which LPS
transduces a signal across the cell membrane remain poorly understood.
Studies with blocking antibodies (3, 4) and transfection experiments
(5, 6) as well as knockout and transgenic models (7-9) have
underscored the importance of CD14, a 55-kDa glycosyl
phosphatidylinositol-linked protein, for enabling cellular responses to
low and intermediate concentrations of LPS. However, CD14 lacks
transmembrane and cytoplasmic regions, and, in addition, high LPS
concentrations elicit cellular activation in CD14 knockout mice (7, 8).
Thus, although CD14 is considered as a component of the LPS receptor
complex, it does not seem to represent a true LPS signal transducing
molecule. Among other LPS binding molecules, complement receptors type
3 and 4 (CR3 and CR4) have been suggested to mediate LPS signaling as
their expression in CHO cells conferred upon them LPS sensitivity, as judged by NF-
B activation (10, 11). However, mutant CR3 molecules that lack cytoplasmic regions also mediate NF-
B activation in response to endotoxin (11). Moreover, monocytes obtained from patients
with a deficiency in expression of CD18, the common
-chain of the
leukocyte integrins, show normal LPS binding and responsiveness (12).
In light of these findings, CD14 and complement receptors have been
proposed to bind LPS and associate with other molecules that mediate
signal transduction. Recently, Yang et al. (13) have
reported that transfection of human embryonic kidney 293 cells with
Toll-like receptor 2 (TLR2) imparts LPS responses that are dependent on
LBP and enhanced by CD14. LPS-induced NF-
B activation has been
demonstrated to require a region in the intracellular domain of TLR2
that is homologous to the intracellular region of the IL-1 receptor
implicated in the activation of the IL-1 receptor-associated kinase
(13). Interestingly, a TLR2 deletion mutant with a truncation of a
region in the intracellular domain appears to act as a
dominant-negative receptor by inhibiting the "endogenous"
LPS-mediated reporter gene activation in U373 astrocytoma cells (13),
further suggesting LPS signaling functions for TLR2. In a related
study, genetic and physical mapping of the mouse Lpsd mutation, which is responsible for LPS
unresponsiveness of C3H/HeJ mice, has revealed a single intact gene
within the entire Lps critical region on mouse chromosome 4 that encodes Toll-like receptor 4 (TLR4) (14). Thus, TLR2 and TLR4,
members of the IL-1 receptor family, are likely to represent LPS
signal-transducing molecules in humans and in the mouse, respectively.
However, further studies will be required to confirm LPS signaling
functions for TLR2 and to demonstrate functional significance of TLR4
in monocytes and macrophages.
Ceramide is an intracellular lipid derived by hydrolysis of
sphingomyelin with sphingomyelin-specific forms of phospholipase C,
neutral and acidic sphingomyelinases (SMase), which is known to mediate
cell activation, proliferation, differentiation, and apoptosis in
response to a number of cytokines and environmental stresses (15-17).
Ceramide activates multiple intracellular targets, including a
ceramide-activated protein kinase (18), identified as the mammalian
homologue of kinase suppressor of Ras (19), a ceramide-activated
protein phosphatase (20), and protein kinase C-
(21). It has been
suggested that LPS may activate cells via its direct stimulation of the
sphingomyelin pathway (22) due to a close structural similarity of the
lipid A portion of the LPS molecule and ceramide (23). Indeed, LPS
activates the ceramide-activated protein kinase in myeloid cells
directly, without stimulation of SMase (23). Phosphorylated
ceramide-activated protein kinase activates Raf-1 (24), which then
phosphorylates and activates the two dual specificity protein kinases,
mitogen-activated protein (MAP) kinase/extracellular signal-regulated
kinase (ERK) kinases 1 and 2, triggering the ERK MAP kinase pathway
(25). Consistent with this scenario, stimulation of the sphingomyelin pathway has been reported to induce ERK activation (26) and NF-
B
translocation (27, 28). However, others have been unable to reproduce
these results (29-32), necessitating further studies on the
involvement of the sphingomyelin pathway in ERK activation and NF-
B
stimulation, responses that are strongly stimulated by LPS (5, 6, 8,
10, 11, 33, 34). Both LPS and ceramides have been found to activate the
c-Jun NH2-terminal kinases (JNK) and the transcription
factor ATF2 (35-37) and to induce the expression of a novel
monocyte/macrophage differentiation-dependent gene (38).
Furthermore, stimulation of C3H/OuJ (Lpsn)
peritoneal macrophages with exogenous SMase or cell-permeable ceramide
analogs activates a subset of LPS-inducible genes, whereas C3H/HeJ
(Lpsd) macrophages fail to respond to either LPS or
ceramide (39, 40). C3H/HeJ (Lpsd) macrophages also
exhibit a defective intracellular transport of LPS and ceramides from
the membrane into the perinuclear region (41), suggesting that this
defect may account for their hyporesponsiveness to both LPS and
ceramide (40). In contrast to LPS, however, cell-permeable ceramide
analogs were poor inducers of interferon-
-inducible protein 10 and
interferon consensus sequence-binding protein gene expression and
interferon secretion in C3H/OuJ (Lpsn) macrophages
(39). In addition, preexposure of macrophages with LPS significantly
decreases TNF release in response to their subsequent stimulation with
LPS, whereas pretreatment of macrophages with SMase does not suppress
LPS-induced TNF production (39). These results suggest the existence of
LPS signal transduction mechanisms that are distinct from the
sphingomyelin pathway.
In the present study, we sought to evaluate further the involvement of
ceramide in LPS-induced macrophage activation. To do so, LPS and
ceramide were compared with respect to their capacities to elicit MAP
kinase phosphorylation, NF-
B and AP-1 transcription factor
activation, and TNF and IL-6 production. The data indicate that
ceramide partially mimics LPS-mediated phosphorylation of the ERK1/2,
JNK1/2, and p38 MAP kinases, as well as AP-1 induction. In contrast to
LPS, ceramide mimetics fail to elicit NF-
B translocation, NF-
B-controlled reporter gene expression, or production of TNF and
IL-6. Strong inhibition of LPS-induced activation of NF-
B and AP-1
was observed by using the LPS structural antagonist, Rhodobacter
sphaeroides diphosphoryl lipid A (RsDPLA), yet RsDPLA did not
antagonize ceramide-induced AP-1 induction. Preexposure of macrophages
with LPS renders them tolerant to subsequent stimulation with either
LPS or C2-ceramide, whereas ceramide treatment does not
induce LPS hyporesponsiveness. Taken together, these results indicate a
limited role of ceramide in LPS-mediated MAP kinase activation,
transcription factor induction, and cytokine production in mouse
macrophages, implying that LPS utilizes both the sphingomyelin pathway
and additional intracellular mechanisms for mediating optimal signal transduction.
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EXPERIMENTAL PROCEDURES |
Reagents--
Protein-free, phenol/water-extracted
Escherichia coli LPS K235 was prepared as described
previously (42). C2-ceramide was purchased from Calbiochem;
C8-ceramide, C2-dihydroceramide, and DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol
(DL-PPMP) were from BIOMOL Research Laboratories, Inc.
(Plymouth Meeting, PA); and SMase from Staphylococcus aureus
was from Sigma. Heat-killed group B streptococcus type III (GBS) was a
gift from Dr. Giuseppe Teti (Institute of Microbiology, University of
Messina, Messina, Italy). The endotoxin content of the preparations was
determined by a Limulus ameobocyte lysate assay and was
0.03-1.5 ng/mg for ceramide preparations, 0.07 ng/mg for
DL-PPMP, 0.5 ng/units for SMase, and 1.4 ng/mg for GBS. The
LPS structural antagonist RsDPLA was prepared as described previously
(43). Recombinant human TNF was kindly provided by Cetus Corp.
(Emeryville, CA) and had a specific activity of 2.7 × 107 units/mg. Rabbit polyclonal antibodies against active
(phosphorylated) ERK1 and -2, JNK1 and -2, and p38
(Anti-activeTM MAP kinase polyclonal antibody) were
generously provided by Dr. Bruce W. Jarvis (Promega). Horseradish
peroxidase-conjugated donkey anti-rabbit IgG was purchased from
Promega. For supershift analyses, antibodies against c-Jun/AP-1 (N)
(sc44x), Jun-B (sc46Gx), Jun-D (sc74x), c-Fos (sc052x), Fos-B (sc48x),
Fra-1 (sc183x), and Fra-2 (sc171x) TransCruzTM were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Tissue Culture--
Peritoneal exudate macrophages were isolated
by peritoneal lavage 3 days after intraperitoneal injection of C3H/OuJ
(Lpsn) or C3H/HeJ (Lpsd) mice
(Jackson Laboratory, Bar Harbor, ME) with 3 ml of sterile 3%
thioglycollate broth. Cells were washed; resuspended in RPMI 1640;
supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES,
0.3% sodium bicarbonate, and 2% FBS; and plated in six-well plates
(5 × 106 cells/well) for preparation of nuclear or
cellular extracts and in 24-well plates (2 × 106
cells/well) for TNF or IL-6 production. To induce cytokine production, macrophages were stimulated with the indicated preparations for 6 h. Supernatants were collected, centrifuged (1,000 × g, 10 min), transferred into sterile Eppendorf tubes, and
stored at
80 °C until use. The mouse WEHI 164 clone 13 fibrosarcoma cell line was generously provided by Dr. Terje Espevik
(Norwegian University of Science and Technology, Trondheim, Norway) and
maintained in RPMI 1640 medium supplemented with 2 mM
L-glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 10% FCS (HyClone) (referred to as RPMI/10% FCS).
The mouse macrophage cell line, RAW 264.7, was obtained from ATCC
(Rockville, MD) and grown in DMEM medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% FCS (referred to as DMEM/10% FCS).
Plasmid Construction and Transient Transfection--
Control
luciferase reporter pLd40-Luciferase (Luc) was constructed
as described previously (44) by cloning the 40-bp H-2Ld
basic promoter into the pGL basic luciferase plasmid (Promega). Reporter plasmids containing an NF-
B enhancer from the murine TNF
promoter 5'-CAAACAGGGGGCTTTCCCTCCTC-3', its mutated version 5'-CAAACAGAGAGCCTTGGCTCCTC-3'
(mutated nucleotides are underlined) (45), and an AP-1 enhancer
5'-CGCTTGATGAGTCAGCCGGAA-3' (Promega) were cloned by inserting three
copies of the respective response elements between the
XhoI-BgIII sites of the pLd40-luciferase
vector. The resulting plasmids were referred to as
p(NF-
B)3LdLuc,
pMut(NF-
B)3LdLuc, and
p(AP-1)3LdLuc. For transient transfections, RAW
264.7 cells were seeded into 12-well plates (Costar) at 2 × 105 cells/well in DMEM/10% FCS, incubated overnight, and
co-transfected for 3 h with the reporter plasmids (0.3 µg/well
of the NF-
B reporters or 0.1 µg/well of the AP-1 reporter
construct) and 0.1 µg/well of pCH110 eukaryotic
-galactosidase
assay vector (Amersham Pharmacia Biotech) by using 7.5 µl/well of
SuperFect transfection reagent (QIAGEN Inc., Chatsworth, CA). The total
amount of plasmid DNA was equalized to 1.5 µg/well by adding
corresponding amounts of pBluescript II SK (+/+) phagemid (Stratagene).
Following transfections, cells were stimulated for 20 h, washed
twice with ice-cold PBS, and lysed in a lysis buffer (Analytical
Luminescence Laboratory, Sparks, MD) for 30 min with constant shaking.
Fifty microliters of the supernatant was assayed in 200 µl of assay
buffer (25 mM glycylglycine, 15 mM
MgSO4, 1% Triton X-100, 1 mM ATP) using a Monolight 2010 luminometer (Analytical Luminescence Laboratory). Following injection of 100 µl of luciferin (0.3 mg/ml, Analytical Luminescence Laboratory), light emission was measured in 10-s intervals. To assay
-galactosidase activity, 20 µl of the
supernatant was mixed with 200 µl of reaction buffer (Galacton-Plus
substrate diluted 1:100 with reaction buffer diluent; Analytical
Luminescence Laboratory), and incubated for 1 h. After injection
of 300 µl of light emission accelerator (Analytical Luminescence
Laboratory), the sample was counted for 5 s. Luciferase activity
was normalized to
-galactosidase activity (normalized relative light
units), as described by Haas et al. (46). Normalized
relative light units obtained in reporter-transfected cells were
divided by those detected in either pMut
(NF-
B)3LdLuc-transfected cells (NF-
B
transactivation) or in pLd40Luc-transfected cells (AP-1
transactivation) for each treatment, respectively, and all data were
normalized to those calculated for medium-treated cells. The resulting
parameter was referred to as "relative fold stimulation" and
reflects the transactivation potential of NF-
B and AP-1.
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared according to Dignam et al. (47) with small
modifications. Briefly, cells were washed twice with ice-cold PBS,
harvested using a rubber policeman, transferred to Eppendorf tubes, and
centrifuged (800 × g, 10 min, 4 °C). Cells were
resuspended in 0.4 ml of ice-cold buffer A (10 mM HEPES, pH
7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, and 1.0 µg/ml each of the following
protease inhibitors: aprotinin, antipain, leupeptin, chymostatin, and
pepstatin), incubated on ice for 15 min, and lysed by the addition of
Nonidet P-40 to a final concentration of 0.5%. Nuclei were pelleted
(1,000 × g, 10 min, 4 °C) and resuspended in 50 µl of ice-cold buffer C (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 25%
glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 µg/ml concentration of the protease inhibitors indicated above). Following a 30-min incubation, the tubes were centrifuged (10,000 × g, 10 min,
4 °C), and supernatants were collected and stored at
80 °C.
Protein concentration was determined by the Bio-Rad protein assay with
bovine serum albumin as a standard (Bio-Rad).
Electrophoretic Mobility Shift Assay
(EMSA)--
NF-
B-specific oligonucleotide
5'-AGTTGAGGGGACTTTCCCAGGC-3' from the murine Ig
B light chain gene
enhancer and AP-1-specific oligonucleotide 5'-CGCTTGATGAGTCAGCCGGAA-3'
probes (synthesized by the BIC Synthesis and Sequencing Facility,
Uniformed Services University of the Health Sciences, Bethesda, MD)
were 32P-end-labeled with T4 polynucleotide kinase
(Promega). Nuclear extracts (5 µg) were incubated with 0.2 ng DNA
probe in a binding buffer containing 2 µg poly (dI-dC) (Amersham
Pharmacia Biotech), 20 mM HEPES, pH 7.9, 50 mM
KCl, 1 mM dithiothreitol, 0.25 mg/ml bovine serum albumin,
4% glycerol for 30 min at room temperature. For supershift assays, 5 µg of nuclear extracts were first preincubated with 0.4 µg of
antibodies against members of the Fos/Jun/Fra family for 45 min at room
temperature in the above mentioned binding buffer. The DNA-protein
complex was resolved from free oligonucleotide by electrophoresis in a
5% polyacrylamide gel (0.25× Tris borate/EDTA, 150 V/2 h). The gels
were dried (80 °C, 2 h) and exposed to x-ray films (X-OMAT AR;
Eastman Kodak Co.).
Preparation of Cellular Extracts and Western
Blotting--
Cellular extracts were obtained as described (48), and
50 µg of total protein was added in Laemmli buffer, boiled for 5 min,
and loaded on SDS-10% polyacrylamide gels for electrophoresis in
Tris/glycine/SDS buffer (25 mM Tris, 250 mM
glycine, 0.1% SDS). Proteins were blotted onto Immobilon-P transfer
membranes (150 V, 1.5 h, 4 °C). The filters were blocked for
20 h at 4 °C in TBS-T (20 mM Tris HCl, 150 mM NaCl, 0.1% Tween 20), containing 1% gelatin and 5%
nonfat milk. Thereafter, the filters were washed three times in TBS-T
and probed for 1 h with anti-phospho-MAP kinase antibodies
(Anti-activeTM antibody; Promega) diluted 1:2,000 in
TBS-T/0.5% nonfat milk, washed three times with TBS-T, and incubated
with secondary horseradish peroxidase-conjugated donkey anti-rabbit
antibody (1:5,000 dilution). The blots were washed 5 times with TBS-T,
and bands were detected by the enhanced chemiluminescence (ECL)
detection method (Amersham Pharmacia Biotech).
TNF and IL-6 Assays--
TNF activity in supernatants was
measured in the WEHI 164 clone 13 bioassay as described previously
(49). The lower limit of detection in this assay was 0.35 pg/ml. IL-6
content of the supernatants was determined on the basis of induction of
proliferation of the IL-6-dependent B.13.29 clone 9 cell
line (generously provided by Dr. T. Espevik) as described elsewhere
(50).
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RESULTS |
Both LPS and Ceramide Cause Phosphorylation of the ERK1/2, JNK1/2,
and p38 MAP Kinases--
To analyze involvement of the sphingomyelin
pathway in LPS signal transduction, the abilities of LPS,
cell-permeable ceramide analogs, and SMase to activate the ERK1/2,
JNK1/2, and p38 MAP kinases in C3H/OuJ (Lpsn)
peritoneal macrophages were compared. A close correlation exists between phosphorylation of the MAP kinases and activation of enzyme activity (51). Therefore, MAP kinase phosphorylation was assessed by
Western blotting, using antibodies specific for phosphorylated forms of
these kinases. Time course experiments revealed that LPS-mediated
phosphorylation of the ERK1/2, JNK1/2, and p38 MAP kinase was evident
after 2-5 min, reached a maximum response within 15-30 min, and
declined by 60 min (Fig. 1). As shown in
Fig. 1, incubation for 15 min was required to permit detectable
phosphorylation of all three MAP kinases in response to SMase or
C2-ceramide, which was significantly lower than that
activated by LPS at this time point. After 30 min of incubation, SMase
and C2-ceramide stimulated maximum levels of MAP kinase
phosphorylation, and the responses decreased by 60 min (Fig. 1). As
little as 1 ng/ml LPS induced phosphorylation of the MAP kinases, and
the minimal effective concentration of SMase was in the range of
31.25-62.5 milliunits/ml, while C2-ceramide was active at
concentrations of
5 µM (data not shown). These results
indicate that although triggering of the sphingomyelin pathway mimics
LPS activation of the ERK1/2, JNK1/2, and p38 MAP kinases
qualitatively, C2-ceramide and SMase mediate delayed and
more transient responses relative to LPS-induced effects.

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Fig. 1.
LPS, C2-ceramide, and SMase
activate the ERK1/2, JNK1/2, and p38 MAP kinases in C3H/OuJ
(Lpsn) mouse macrophages. Cells were
stimulated for the indicated time periods with 100 ng/ml LPS, 50 µM C2-ceramide, and 250 milliunits/ml SMase.
Phosphorylation of the ERK1/2, JNK1/2, and p38 MAP kinase was measured
in total cellular extracts by Western blotting with anti-phospho-MAP
kinase antibodies as described under "Experimental Procedures." The
results of a representative experiment (n = 3) are
presented.
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LPS, but Not C2-ceramide or SMase Induces NF-
B
Activation--
The ability of LPS and ceramide to activate the
transcription factor NF-
B was next examined by measuring NF-
B DNA
binding and transcriptional activities. Fig.
2A demonstrates that LPS induced NF-
B translocation in C3H/OuJ (Lpsn)
macrophages after 15 min and exerted a maximal effect at 30-60 min,
and the response decreased by 180 min of stimulation. Dose-response experiments showed evident NF-
B translocation caused by 0.1 ng/ml LPS, which reached a plateau at 1-1,000 ng/ml LPS (data not shown). Endotoxin-hyporesponsive C3H/HeJ (Lpsd) macrophages
did not exhibit NF-
B activation following LPS treatment (Fig.
2A). In contrast to LPS, neither C2-ceramide
(Fig. 2A) nor C8- and C6-ceramide
analogs (data not shown) led to NF-
B translocation in macrophages
from either mouse strain throughout 180 min of stimulation. Similarly,
no NF-
B translocation was seen after macrophage treatment with SMase
or DL-PPMP (data not shown), agents that increase
intracellular concentrations of endogenous ceramide. When Gram-positive
heat-killed GBS was used as a stimulus, similar patterns of NF-
B
induction were observed in macrophages from both mouse strains (Fig.
2A), ruling out the possibility of generalized hyporeactivity of C3H/HeJ cells. To extend the EMSA data, we also studied the capacities of LPS and ceramide mimetics to activate NF-
B-dependent transcription in RAW 264.7 cells
transiently transfected with the
p(NF-
B)3LdLuc reporter construct. As
depicted in Fig. 2B, 100 ng/ml LPS potently activated
NF-
B-dependent Luc gene expression (relative fold
stimulation compared with medium-treated cells is 15.0 ± 1.5).
Ten and 1 ng/ml LPS caused 10 ± 1.2 and 7.7 ± 2.1 stimulation of the NF-
B reporter, respectively, whereas 0.1 ng/ml
LPS caused a 2-fold stimulation. Importantly, LPS did not activate the
construct with a mutated NF-
B consensus sequence, indicating that
the response was NF-
B-specific. Similar to the failure to cause
NF-
B translocation in mouse macrophages (Fig. 2A) and in
RAW 264.7 cells (data not shown), neither C2-ceramide,
SMase, nor DL-PPMP activated NF-
B-dependent transcription (Fig. 2B). Thus, in contrast to LPS,
triggering of the sphingomyelin pathway does not activate NF-
B in
mouse macrophages.

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Fig. 2.
Effect of LPS and agents that trigger the
sphingomyelin pathway on NF- B DNA binding and
transcriptional activities in mouse macrophages. A,
C3H/OuJ (Lpsn) or C3H/HeJ (Lpsd)
macrophages were treated for the indicated periods of time with 1 µg/ml LPS, 50 µM C2-ceramide, and 50 µg/ml GBS. Nuclear extracts were prepared and analyzed by EMSA. The
data of a representative experiment (n = 6) are shown.
B, RAW 264.7 macrophages were transiently co-transfected
with 0.3 µg of p(NF- B)3LdLuc or 0.3 µg
of Mut p(NF- B)3LdLuc, along with 0.1 µg of
pCH110, followed by stimulation for 20 h with indicated
concentrations of LPS, SMase, DL-PPMP, and
C2-ceramide. Lysates were collected and assayed for
luciferase and -galactosidase activities. Data (mean ± S.E.,
n = 4) are presented as relative fold stimulation
compared with medium-treated cells.
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|
Both LPS and Ceramide Activate the Transcription Factor
AP-1--
To evaluate further if LPS signals cell activation by
mimicking ceramide, we compared the effects of LPS,
C2-ceramide, SMase, and DL-PPMP on AP-1
activation. Treatment of C3H/OuJ macrophages with LPS for 30 min
resulted in an increased AP-1 DNA binding, which reached a plateau by
60-120 min and declined by 180 min post-stimulation (Fig.
3A). As demonstrated in Fig.
3, A and B, SMase, C2-ceramide, and
DL-PPMP also activated AP-1 DNA binding but with slower
kinetics compared with that induced by LPS. As little as 0.01 ng/ml LPS
induced AP-1 transcriptional activity by 2.5-fold in RAW 264.7 cells
transiently transfected with the p(AP-1)3LdLuc
reporter plasmid, whereas 1-100 ng/ml LPS caused a stimulation up to
6.7-fold (Fig. 3C and data not shown). Although less potent than LPS, SMase, DL-PPMP, and C2-ceramide all
resulted in the induction of AP-1-dependent reporter gene
expression in a dose-dependent fashion (Fig.
3C). The inactive ceramide analog,
C2-dihydroceramide, did not activate AP-1 (Fig.
3B). Neither LPS nor C2-ceramide stimulated AP-1
DNA binding in LPS-hyporesponsive C3H/HeJ mouse macrophages (Fig. 3),
supporting a correlation between LPS hyposensitivity and defective
ceramide responses described previously at the level of gene expression
(39, 40). These results demonstrate that ceramide partially mimics
LPS-mediated activation of AP-1 DNA-binding and transcriptional
activities, exhibiting delayed and less potent responses compared with
LPS-mediated effects.

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Fig. 3.
Effect of LPS and ceramide mimetics on AP-1
DNA-binding and transcriptional activities. A, C3H/OuJ
(Lpsn) and C3H/HeJ (Lpsd)
macrophages were stimulated for various time periods with either 1 µg/ml LPS or 50 µM C2-ceramide.
B, 250 milliunits/ml SMase, 10 µM
DL-PPMP, or 50 µM C2-dihydroceramide were
added to C3H/OuJ (Lpsn) macrophages, and incubation
continued until the indicated time points. Nuclear extracts were
prepared and assayed for AP-1 induction by EMSA. Shown are the results
of a representative experiment (n = 3). C,
RAW 264.7 cells were transiently co-transfected with 0.1 µg of pCH110
and either p(AP-1)3LdLuc or pLdLuc
(0.1 µg each). After stimulation with the indicated concentrations of
LPS, SMase, DL-PPMP, or C2-ceramide, lysates
were assayed for luciferase and -galactosidase activities, and data
(mean ± S.E., n = 3) are expressed as described
in the legend to Fig. 2.
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Different Members of the Jun/Fos Family Comprise AP-1 Complexes
Induced by LPS Versus C2-ceramide--
The
trans-activating potential of many transcription factors, including
AP-1, is determined by their subunit composition (52). Hence, it was of
interest to examine whether different potencies of LPS and ceramide in
mediating AP-1 trans-activation correlate with different subunit
compositions of AP-1 complexes induced by these stimuli. To this end,
we performed supershift analyses of nuclear extracts obtained from LPS-
and C2-ceramide-stimulated macrophages using antibodies
against members of the Fos/Jun family. Fig.
4 shows that the addition of antibodies
against c-Fos, Jun-B, c-Jun, and Jun-D resulted in the appearance of
slower migrating species relative to the major LPS-inducible AP-1 band,
whereas anti-Fos-B antibodies had no effect. In contrast, only c-Jun
and Jun-D comprised AP-1 complexes induced by C2-ceramide
(Fig. 4). Antibodies against Fra-1 and Fra-2 did not change the
electrophoretic mobility of the AP-1 band in samples from either LPS-
or C2-ceramide-stimulated macrophages (Fig. 4). AP-1-DNA
complexes induced by either stimulus were reduced in the presence of an
excess of unlabeled AP-1, but not NF-
B, consensus oligonucleotides
(data not shown), demonstrating the specificity of the response. Taken
collectively, these data demonstrate that LPS stimulates both Fos and
Jun members of the AP-1 superfamily, while only c-Jun and Jun-D are
activated by C2-ceramide.

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Fig. 4.
Supershift analyses of AP-1 complexes induced
by LPS and C2-ceramide. C3H/OuJ macrophages were
treated for 2 h with medium, 1 µg/ml LPS (top panel),
or 50 µM C2-ceramide (bottom
panel). Nuclear extracts were preincubated for 45 min at room
temperature with the indicated antibodies against members of the
Jun/Fos superfamily, and AP-1 DNA binding was analyzed by EMSA. The
results of a representative experiment (n = 2) are
presented.
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|
RsDPLA Inhibits LPS-induced NF-
B and AP-1 Activation but Does
Not Affect C2-ceramide-mediated AP-1 Response--
In the
next series of experiments, the effect of the LPS antagonist, RsDPLA,
on transcription factor induction induced by LPS and
C2-ceramide was evaluated. Pretreatment of C3H/OuJ
macrophages with 10 µg/ml RsDPLA significantly decreased both NF-
B
and AP-1 activation caused by 0.1-100 ng/ml LPS, whereas 1,000 ng/ml
LPS overcame the inhibitory effect of RsDPLA (Fig.
5, A and B).
Neither NF-
B nor AP-1 were induced by RsDPLA alone. Ten, 5, and 2.5 µg/ml RsDPLA completely abrogated NF-
B and AP-1 activation in
response to 1 ng/ml LPS, whereas no inhibition was observed with
concentrations of RsDPLA of <0.32 µg/ml (data not shown). In
contrast to its potent inhibition of LPS-induced responses, RsDPLA
did not affect C2-ceramide-mediated AP-1 activation, even
when used at 10 µg/ml (Fig. 5C). Similar data were
obtained with C8-ceramide (data not shown), demonstrating
that RsDPLA fails to inhibit ceramide-induced AP-1 induction.

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Fig. 5.
RsDPLA inhibits LPS-induced activation of
transcription factors NF- B and AP-1, whereas it does not affect
C2-ceramide-mediated AP-1 response. C3H/OuJ peritoneal
macrophages were pretreated with 10 µg/ml of RsDPLA or medium for 30 min, followed by the addition of LPS or C2-ceramide as
indicated. After cells were stimulated for 60 min (A) or 120 min (B and C), nuclear extracts were prepared and
assayed for NF- B (A) or AP-1 (B) in
EMSA.
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The Addition of C2-ceramide or SMase Does Not Affect
LPS-induced NF-
B Activation and Cytokine Production but Enhances the
LPS-mediated AP-1 Response--
The next goal of the study was to
elucidate if triggering of the sphingomyelin pathway modulates
LPS-initiated responses. C3H/OuJ macrophages were stimulated with
suboptimal LPS concentrations in the absence or presence of
C2-ceramide, and NF-
B and AP-1 activation as well as TNF
and IL-6 production were measured as functional parameters of LPS
signaling. Treatment of cells with 1 ng/ml LPS for 30 min led to a
marked NF-
B translocation, whereas C2-ceramide neither
induced NF-
B nor affected the LPS response (Fig.
6). Similar data were obtained when LPS
was used at concentrations of 0.1 and 10 ng/ml and when NF-
B
translocation was assessed throughout a 15-120-min period of
stimulation (data not shown). Interestingly, LPS or
C2-ceramide alone activated AP-1 DNA binding, and their
combination gave rise to an additive response (Fig. 6). Stimulation of
macrophages with 0.1 and 1 ng/ml LPS for 6 h resulted in the
production of TNF (1,000 ± 150 and 10,300 ± 689 pg/ml) and
IL-6 (1,100 ± 75 and 5,475 ± 487 pg/ml), respectively. In
contrast, C2-ceramide used at concentrations of 1, 12.5, and 25 µM did not stimulate the release of TNF or IL-6
above levels seen in medium-treated macrophages (1 ± 0.3 pg/ml
TNF and 41 ± 6 pg/ml IL-6). Consistent with the NF-
B data
(Fig. 6), simultaneous addition of 1, 12.5, and 25 µM
C2-ceramide did not modulate LPS-induced TNF and IL-6
responses. The inactive ceramide analogue,
C2-dihydroceramide, had no effect on LPS-mediated
transcription factor activation and cytokine production (data not
shown). These results indicate that C2-ceramide-induced
signal transduction pathways do not contribute to LPS-induced NF-
B
activation and production of TNF and IL-6. On the other hand, AP-1
stimulation induced by LPS and C2-ceramide may be triggered
by distinct mechanisms that converge downstream, leading to an additive
effect.

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Fig. 6.
Effect of C2-ceramide addition on
LPS-mediated induction of NF- B and AP-1.
C3H/OuJ macrophages were treated for 60 min with 1 ng/ml LPS in the
absence or presence of 25 µM C2-ceramide.
NF- B and AP-1 DNA binding activities were measured in nuclear
extracts by EMSA, as described under "Experimental
Procedures."
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LPS Pretreatment Inhibits Macrophage Activation by LPS and
C2-ceramide, whereas C2-ceramide Enables
Macrophage Tolerance to C2-ceramide but Not to
LPS--
Next, a model of macrophage tolerance to LPS in
vitro (53, 54) was utilized to examine the potential role of
ceramide in LPS-mediated transcription factor activation and cytokine
production. First, C3H/OuJ macrophages were pretreated for 20 h
with 10 ng/ml LPS, washed, rested in fresh medium for 2 h, and
restimulated with either LPS or C2-ceramide. LPS
pretreatment significantly decreased the ability of macrophages to
induce NF-
B and AP-1 (Fig. 7) as well
as to secrete TNF and IL-6 (Fig. 8) in
response to subsequent stimulation with LPS. Likewise, prior exposure
of macrophages to LPS markedly inhibited their AP-1 response induced by
C2-ceramide (Fig. 7). In contrast, preincubation of
macrophages with C2-ceramide did not affect LPS-mediated
NF-
B translocation, AP-1 induction (Fig. 7), TNF, or IL-6 production
(Fig. 8, A and B), while it completely abolished
AP-1 induction in response to C2-ceramide (Fig. 7). Similar
results were obtained when SMase was used to generate intracellular
ceramide (data not shown). Thus, prior exposure of macrophages with
ceramide mimetics results in down-modulation of the sphingomyelin
pathway, inhibiting subsequent induction of an AP-1 response by
ceramide, whereas LPS-mediated transcription factor activation and
cytokine production remain unaffected. In contrast, LPS pretreatment
renders macrophages unresponsive to both LPS and ceramide.

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Fig. 7.
Comparison of the abilities of LPS and
C2-ceramide to induce macrophage
NF- B and AP-1 hyporesponsiveness in response
to subsequent stimulation with LPS and C2-ceramide.
C3H/OuJ macrophages were pretreated for 20 h with medium, 10 ng/ml
of LPS, 50 µM C2-ceramide. Thereafter, cells
were washed three times with medium, rested in medium for 2 h,
washed again, and stimulated with 100 ng/ml of LPS or 50 µM C2-ceramide for the indicated periods of
time. Nuclear extracts were prepared and assayed for NF- B and AP-1
DNA binding by EMSA. Presented are the results of a representative
experiment (n = 4).
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Fig. 8.
Effect of macrophage pretreatment with LPS or
C2-ceramide on TNF and IL-6 production after subsequent LPS
challenge. C3H/OuJ macrophages were pretreated for 20 h with
medium, 10 ng/ml of LPS, 10 µM C2-ceramide,
or 10 µM C2-dihydroceramide. After
washing, cells were rested in fresh medium for 2 h, washed, and
stimulated with a serial dilution of LPS. Following incubation for
6 h, cell-free supernatants were collected and analyzed for TNF
(A) and IL-6 (B). The data (mean ± S.D.) of
a representative experiment (n = 4) are shown.
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DISCUSSION |
LPS has been proposed to signal cell activation by mimicking the
intracellular second messenger ceramide (23), due to a close structural
similarity between the lipid A portion of LPS and the ceramide molecule
(24). One implication of this model is that similar intracellular
pathways are engaged by LPS and ceramide that consequently lead to
overlapping responses. This paper demonstrates the failure of
LPS-hyporesponsive C3H/HeJ macrophages to exhibit NF-
B and AP-1
activation following stimulation with LPS, SMase, and
C2-ceramide. Our results extend the correlation between LPS
and ceramide hyporesponsiveness of C3H/HeJ macrophages found previously
at the level of gene expression (40) and support involvement of the
sphingomyelin pathway in LPS signaling. However, several observations
shown in the present study strongly suggest that activation of the
sphingomyelin pathway is one of multiple signals initiated by LPS in
mouse macrophages. First, LPS and ceramide were found to exhibit
qualitative differences in the elicitation of several cellular
responses. Indeed, in contrast to LPS, cell-permeable ceramide analogs,
DL-PPMP, and SMase did not stimulate NF-
B translocation
in C3H/OuJ macrophages and failed to trigger
NF-
B-dependent transcription of the Luc reporter gene in
RAW 264.7 cells. These data support and extend earlier publications on
the inability of ceramide to induce NF-
B found in other cell types
(30-32, 36). However, conflicting reports exist that demonstrate the
capacities of ceramide analogs to activate NF-
B in HL60
promyelocytic and EL4 lymphoma cells (27, 28) and to potentiate TNF- or LPS-induced NF-
B responses in human vein endothelial cells and in
primary rat astrocytes (55, 56). Therefore, to determine unequivocally
the role of ceramide in NF-
B activation in mouse macrophages, we
analyzed how independent triggering and down-modulation of the
sphingomyelin pathway affect LPS-initiated NF-
B translocation. Not
only did C2-ceramide lack the ability to activate NF-
B
by itself, but also it failed to influence LPS-initiated NF-
B
responses when present in the cell cultures simultaneously with LPS.
Furthermore, pretreatment of macrophages with C2-ceramide
did not affect LPS-mediated NF-
B translocation, while it completely
abolished subsequent induction of AP-1 by ceramide mimetics. In
contrast, LPS pretreatment rendered macrophages hyporesponsive to
subsequent challenge with either LPS or ceramide mimetics. These data
demonstrate that ceramide-elicited refractoriness of macrophages to
itself has no effect on the ability of LPS to activate NF-
B. Taken
together, these results indicate that, in mouse macrophages, ceramide
is not involved in LPS-mediated NF-
B activation. Similar to the
NF-
B data, LPS, but not C2-ceramide, stimulated C3H/OuJ
macrophages to secrete TNF and IL-6. Moreover, no modulatory effect of
C2-ceramide on LPS-mediated cytokine release was observed
when these reagents were added simultaneously or when cells were first
pretreated with C2-ceramide. Thus, at least three LPS
responses exist, i.e. NF-
B activation as well as TNF and
IL-6 release, that could not be induced or modulated by ceramide mimetics.
Second, to delineate whether LPS and ceramide involve similar
mechanisms for transcription factor activation, we employed the LPS
antagonist, RsDPLA (54, 57). Importantly, RsDPLA, as well as other LPS
partial structures, e.g. deacylated LPS or lipid
IVA, suppress LPS activities at concentrations that do not affect LPS binding to CD14 (58), suggesting that they compete with LPS
for a putative signaling receptor distinct from CD14 (58). An
alternative hypothesis postulates that RsDPLA could trigger negative
intracellular events that interfere with LPS signaling further
downstream at the postreceptor level. Thus far, however, no
evidence exists to support the latter model, since RsDPLA fails to
induce transcription factor activation (Fig. 4), MAP kinase
phosphorylation, or cytokine release (data not shown). Regardless of
the exact mechanism of action of RsDPLA, we reasoned that if
LPS-mediated macrophage activation occurs via the sphingomyelin pathway, then RsDPLA should inhibit both LPS- and ceramide-mediated effects. However, this is not the case, as treatment of C3H/OuJ macrophages with RsDPLA did not affect AP-1 activation in response to
C2-ceramide, whereas this compound significantly inhibited LPS-induced NF-
B and AP-1. Thus, RsDPLA-inhibitable molecules engaged in LPS-mediated transcription factor activation do not represent ceramide-triggered signaling components.
Third, this paper shows that even when overlapping responses are
elicited, LPS and ceramide seem to involve divergent intracellular mechanisms. Both LPS and ceramide mediated AP-1 activation; however, different kinetics of AP-1 induction, subunit compositions of AP-1
complexes, and different potencies of AP-1 trans-activation were seen
in response to LPS versus ceramide. Furthermore, an additive
effect on AP-1 DNA-binding activity was observed when macrophages were
stimulated with a combination of LPS and C2-ceramide. Finally, macrophages pretreated with LPS manifested a significantly lower AP-1 response upon subsequent stimulation with LPS,
C2-ceramide, or SMase, whereas prior exposure to
C2-ceramide conferred tolerance only to
C2-ceramide. Our results extend earlier published
observations on the ability of the sphingomyelin pathway to signal AP-1
activation in the human leukemia cell line HL-60 (59) and in the
insulin-producing cell line RINm5F (36), studies where a comparative
analysis of LPS versus ceramide responses was not
undertaken. Furthermore, they suggest that quantitative differences
between LPS and ceramide with respect to AP-1 trans-activation are
likely to reflect their capacities to stimulate AP-1 complexes with
different subunit compositions. Indeed, both Fos and Jun proteins
comprised LPS-induced AP-1, which have been reported to form more
stable complexes and activate transcription more efficiently than Jun
subunits (60, 61), whose up-regulation was caused by
C2-ceramide.
Analysis of MAP kinase phosphorylation also underscored the kinetic
differences between LPS and ceramide mimetics. To the best of our
knowledge, this paper shows for the first time that ceramide is capable
of mediating phosphorylation of p38 MAP kinase in mouse macrophages,
extending a similar observation made recently in Jurkat cells (62). In
addition, we have found that LPS, C2-ceramide, or SMase
shared the ability to mediate phosphorylation of ERK1/2 MAP kinases, an
observation for which conflicting data have been reported (26, 29). In
agreement with earlier results (32-35), C2-ceramide or
SMase also mimicked LPS-induced phosphorylation of the JNK1 and two MAP
kinases. However, despite the fact that both LPS,
C2-ceramide, and SMase caused phosphorylation of ERK1/2, JNK1/2, and p38 MAP kinases, slower and less sustained responses were
induced by ceramide mimetics compared with LPS. It is tempting to
speculate that differential activation of protein phosphatases by LPS
versus ceramide accounts for this phenomenon, limiting MAP
kinase-mediated downstream signaling triggered by ceramide. Since
different patterns of cellular responses could be induced depending on
whether transient or sustained MAP kinase activation occurs (63),
this could have functional consequences for differential effect of LPS
and ceramide on both transcription factor activation and cytokine
production. Experiments are in progress to examine this hypothesis.
NF-
B is a prerequisite for the expression of many cytokine genes,
including TNF (45), IL-6 (64), granulocyte-macrophage colony-stimulating factor (65), and interferon-
-inducible protein 10 (66). Therefore, the failure of ceramide to activate NF-
B is likely
to reflect its inability to elicit TNF and IL-6 production (data
herein, and see Refs. 67-69) and to induce interferon-
-inducible protein 10 gene expression (39). In addition, recent reverse transcription-polymerase chain reaction analyses of cytokine gene expression in C3H/OuJ macrophages have demonstrated that SMase and
cell-permeable ceramide analogs are very poor inducers of the
expression of mRNA for another NF-
B-dependent gene,
granulocyte-macrophage colony-stimulating
factor.2 Furthermore,
ceramide is incapable of activating C3H/OuJ macrophages to secrete
nitric oxide (70), a process that requires NF-
B activation for the
induction of inducible nitric-oxide synthase mRNA (71). These
results are consistent with recent findings in which unimpaired
cytokine production in acid SMase knock-out mice (68, 69) as well as
normal NF-
B activation in acid SMase-deficient cells (32) have been
demonstrated. On the other hand, stimulation of the sphingomyelin
pathway with cell-permeable ceramide analogs (39) or SMase (data not
shown) mimics LPS-induced expression of IL-1
mRNA in mouse
C3H/OuJ macrophages. It is important to note that in mice, IL-1
gene
expression is NF-
B-independent but is controlled by several
transcription factors, including AP-1, NF-IL6, and PU-1 (72-74).
Therefore, it is plausible that ceramide-induced AP-1 activation
contributes to stimulation of IL-1
gene expression in our system.
More generally, it suggests a functional role of the sphingomyelin
pathway in mediating expression of NF-
B-independent genes, whose
stimulation requires AP-1. Another important consequence of AP-1
activation by ceramide underlies apoptotic death of human leukemia
HL-60 cells (59), implying that ceramide-induced cell death of C3H/OuJ
mouse macrophages (70) could also be AP-1-mediated. However, C3H/OuJ
macrophages exhibit only necrotic cell death in response to ceramide
analogs and mimetics, whereas LPS induces both necrosis and apoptosis (70), again suggesting the existence of additional intracellular pathways activated by LPS.
In summary, several novel findings presented herein indicate that LPS
involves not only the sphingomyelin pathway but also alternate
intracellular pathways to evoke MAP kinase activation, transcription
factor induction, and production of TNF and IL-6 in mouse macrophages.
These include the lack of capacity of ceramide to mimic LPS-mediated
NF-
B activation, TNF, and IL-6 production, its failure to tolerize
macrophages against LPS, and the ability of RsDPLA to inhibit
LPS-induced transcription factor activation without affecting
ceramide-induced AP-1. Furthermore, even in the case where a certain
response, e.g. AP-1 activation, is elicited by both LPS and
ceramide, they are likely to utilize distinct intracellular pathways.
Identification of upstream regulators of MAP kinase cascades triggered
by LPS and ceramide as well as characterization of their downstream
targets (e.g. kinases and transcription factors) will help
bring about a better understanding of the contribution of the
sphingomyelin pathway to LPS signal transduction.