From the Evans Biomedical Research Center, Boston
Medical Center, Boston, Massachusetts 02118, the
§ Institute of Cancer Research and Molecular Biology,
Norwegian University of Science and Technology,
7489 Trondheim, Norway,
Eisai Research Institute,
Andover, Massachusetts 01810, and ** Saga Medical School,
Saga 849-8501, Japan
Received for publication, October 3, 2000, and in revised form, October 12, 2000
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ABSTRACT |
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ER-112022 is a novel acyclic synthetic lipid A
analog that contains six symmetrically organized fatty acids on a
noncarbohydrate backbone. Chinese hamster ovary (CHO)-K1 fibroblasts
and U373 human astrocytoma cells do not respond to lipopolysaccharide
(LPS) in the absence of CD14. In contrast, exposure to ER-112022
effectively induced activation of CHO and U373 cells under serum-free
conditions. Expression of CD14 was not necessary for cells to respond
to ER-112022, although the presence of soluble CD14 enhanced the
sensitivity of the response. Several lines of evidence suggested that
ER-112022 stimulates cells via the LPS signal transduction pathway.
First, the diglucosamine-based LPS antagonists E5564 and E5531 blocked ER-112022-induced stimulation of CHO-K1, U373, and RAW264.7 cells. Second, ER-112022 was unable to activate C3H/HeJ mouse peritoneal macrophages, containing a mutation in Toll-like receptor (TLR) 4, as
well as HEK293 cells, an epithelial cell line that does not express
TLR4. Third, ER-112022 activated NF- Lipopolysaccharide
(LPS,1 endotoxin) is the
major constituent of the outer envelope of Gram-negative bacteria. LPS
is thought to be a major cause of the widespread cellular activation
observed in patients with Gram-negative septic shock. The lipid A
portion of LPS consists of a backbone of two phosphorylated glucosamine molecules acylated with fatty acids and contains the pro-inflammatory toxicity associated with LPS (1, 2). LPS activation of mammalian cells
is mediated by its interaction with membrane CD14, a 55-kDa glycosylphosphatidylinositol (GPI)-anchored protein primarily expressed
on phagocytes (3, 4). CD14 appears to be the highest affinity receptor
for LPS, but other LPS-binding molecules involved in proinflammatory
responses by phagocytic leukocytes have been described, such as the
Several lines of evidence suggest that TLR4 is the main
signal-transducing molecule in the LPS receptor complex. First, animals that lack a functional TLR4, but not those without a functional TLR2,
are markedly hyporesponsive to LPS (11, 18-22). Second, LPS induces
activation of HEK293 cells transfected with TLR4 cDNA, and this
activation is blocked by a lipid A-based LPS antagonist (12). Third,
TLR4 is responsible for the fine discriminatory ability of the LPS
receptor. For example, the unique species-specific pharmacology
associated with small alterations in lipid A structure can be
attributed to differences in the primary structure of TLR4 (23, 24). In
contrast, TLR2 appears to be involved in responses to a variety of
bacterial compounds, such as bacterial lipoproteins, peptidoglycan, and
lipoarabinomannan, as well as whole bacteria and yeast particles (21,
22, 25-34). It is still unclear whether TLR2 can mediate lipid A
signals (35), although it is possible that TLR2 represents an LPS
receptor involved in the recognition of non-enteric endotoxins.
An important approach to understanding how any receptor system
functions is to define its pharmacology. Partial lipid A structures have been important in investigating mechanisms of LPS binding and cell
activation (36). These compounds include several naturally occurring,
bacterially derived LPS antagonists such as deacylated LPS, lipid IVa,
and Rhodobacter sphaeroides lipid A (37-39) as well as
several synthetic lipid A-like structures (40-42) that do not exist in
nature. One advantage of the synthetic compounds is that they are less
likely to be contaminated with minor, but potent, bacterial products
that may mislead the investigator, as has been an occasional
confounding problem with LPS (35, 43).
In an attempt to dissect further the LPS-signaling pathway, we utilized
a newly developed lipid A agonist, compound ER-112022. This compound is
a symmetric phospholipid dimer connected by a noncarbohydrate backbone.
We observed that under serum-free conditions, ER-112022 was able to
stimulate cells lacking membrane CD14. Furthermore, ER-112022 appeared
to mediate stimulatory effects through the TLR4 and MD-2 pathway. We
suggest that ER-112022 interacts directly with the LPS-signaling
receptor, obviating the need for CD14.
Reagents--
PBS, trypsin-EDTA, DMEM (high glucose), and Ham's
F12 medium were obtained from BioWhittaker (Walkerswille, MD); Ex-Cell
301 serum-free medium was from JRH Biosciences (Lenexa, KS); and
Hanks' balanced salt solution, glutamine, and RPMI 1640 were from Life Technologies, Inc. Fetal calf serum (FCS) was from Summit
Biotechnologies (Greeley, CO). Human serum was collected in Vacutainer
SST gel and clot activator tubes (Becton Dickinson, San Jose, CA),
separated from cellular elements by centrifugation at 4 °C, and
stored at
Synthetic lipid A-like compounds used in this study,
ER-112022 (53), and the LPS antagonists E5564 (also known
as B1287) (41, 42) and E5531 (40) were synthesized at the Eisai
Research Institute (Andover, MA). All lipids (including LPS) were
stored as frozen stock suspensions in PBS at 1 mg/ml. Lipids were
thawed and sonicated 1-3 min before use. The activity of compound
ER-112022 in the Limulus amebocyte lysate assay
(Chromogenix, Mölndal, Sweden) was less than 0.3 pg/µg.
Cells and Cell Culture Conditions--
The Chinese hamster ovary
(CHO)-K1 fibroblast cell line, the mouse macrophage cell line RAW264.7,
and the human astrocytoma U373 cell line were obtained from the
American Type Culture Collection (Manassas, VA). CHO cells were grown
in Ham's F12 medium containing 10% fetal bovine serum and
ciprofloxacin (10 µg/ml), whereas RAW and U373 were maintained in
DMEM containing 10% FCS, L-glutamine, and
penicillin/streptomycin (Life Technologies, Inc.). The stably transfected cell lines CHO/CD14 and CHO/Neo (44) were grown in medium
containing 0.5 mg of G418/ml (Life Technologies, Inc.). The reporter
cell lines CHO/CD14/Elam.Tac (clone 3E10) (45) and CHO/Elam.Tac were
cultured in complete medium supplemented with 400 units of hygromycin
B/ml (Calbiochem).
The NF-
The day before each stimulation experiment, CHO cells were washed once
with PBS, harvested using trypsin-EDTA, and plated in 6-well dishes
(Costar) at densities of 0.5 × 106/well (for
preparation of nuclear extracts) or 1 × 106/well (for
flow cytometry analysis) in 1 ml of fresh culture medium. Immediately
prior to stimulation, cells were washed three times with PBS, and the
indicated medium was added. When analyzing the effect of the presence
of serum or serum components, cells were stimulated in Ex-Cell
serum-free medium with or without additives (human serum, sCD14, and
LBP).
U373 cells were seeded in 96-well dishes at a density of 2 × 104 cells/well. The following day, the cells were washed
three times with Hanks' balanced salt solution (Life Technologies,
Inc.), and the indicated medium was added. Cells were stimulated for 16-18 h, and supernatants were analyzed for IL-6 content by ELISA (Endogen, Woburn, MA).
RAW cells were plated at a density of 2 × 105
cells/well in 96-well plates. The following day, the cells were washed
three times with serum-free DMEM, and the indicated medium was added. Cells were stimulated for 3 h, and the TNF content of the
supernatants was determined using a commercial ELISA kit (Genzyme,
Cambridge, MA).
Freshly isolated adherent human monocytes were isolated as described
previously (46). The cells were stimulated in RPMI medium without
serum, in the presence or absence of 10 µg/ml of the following
antibodies: 6H8 control antibody against a widely distributed myeloid
antigen,2 the TLR2 antibody
TL2.1 (31), or the TLR4 antibody HTA125 (13). After 8 h of
stimulation, cell-free supernatants were harvested and subsequently
analyzed for TNF by the WEHI 164 clone 13 bioassay (47).
Transient Transfection--
The human embryonic kidney
epithelial cell line HEK293 was originally obtained from ATCC and
maintained in DMEM containing 10% FCS. The day before the experiment,
the cells were plated at a density of 3 × 105/well in
24-well dishes. Cells were transfected by the calcium phosphate method
(CalPhos, CLONTECH, Palo Alto, CA) with the
following plasmids: TLR4 in the vector pcDNA3 (a gift from R. Medzhitov and C. Janeway, Yale University), MD-2 in the vector pEF-BOS
(13), or the empty vector pcDNA3 (Invitrogen, Carlsbad, CA). The
total amount of plasmid was kept at a constant 90 ng by the addition of
empty vector. In addition, all cells were co-transfected with 50 ng of
the NF- Peritoneal Macrophages--
Peritoneal macrophages from C3H/HeN
and C3H/HeJ mice (Harlan Ltd., Oxon, UK) were collected by lavage 4 days after intraperitoneal injection of 3% thioglycollate (3 ml,
Difco). Cells (5 × 105 cells/well in 24-well dishes)
were adhered for 2 h, and nonadherent cells were removed by
washing three times with PBS. Macrophages were stimulated in RPMI, 10%
FCS for 8 h in a total volume of 0.25 ml. Cell-free supernatants
were analyzed for TNF by the WEHI 164 clone 13 bioassay (47).
Flow Cytometry Analysis (FACS) of NF- Preparation of Nuclear Extracts and NF- Structure of Compound ER-112022--
Several recent advances in
the chemical synthesis of compounds are notable because they provide
tools for characterizing the LPS signal transduction system. Both E5531
(40) and E5564 (41, 42) (Fig. 1) are
potent LPS antagonists that are under development as therapeutic
compounds for the treatment of sepsis. In contrast, compound ER-112022
(Fig. 1) has no LPS antagonistic activity but rather is an LPS mimetic.
ER-112022 is a phospholipid dimer connected via an acyclic backbone.
Each monomeric unit contains three unique fat groups that are bound
indirectly to a phosphate diester. The fat groups include a 10-carbon
ether chain, an unsaturated 12-carbon, acyloxy chain bound to the ether
chain, and a 14-carbon, ER-112022 Stimulates CHO Cells Bypassing CD14 in a
Serum-independent Fashion--
We added ER-112022 to CHO fibroblasts
transfected with human CD14 (CHO/CD14) and measured nuclear
translocation of NF- Soluble CD14, but Not LBP, Enhances the Activity of
ER-112022--
To identify the serum proteins that enhanced the
stimulatory effects of ER-112022 on CHO cells, CHO/Elam.Tac NF-
We further analyzed the effect of serum and sCD14 on the cellular
activation induced by ER-112022 in other cell lines, such as U373
astrocytoma cells and RAW264.7 monocytes/macrophages. Both of these
lines differ from CHO cells in their requirements for serum proteins to
respond to LPS; for example, the CD14-negative U373 cell line is
enabled to respond to LPS in the presence of sCD14 (16), and the RAW
cell line expresses GPI-linked CD14. U373 cells responded to LPS to
release IL-6 only in the presence of serum or sCD14 (Fig.
4A). In contrast to LPS,
ER-112022 induced IL-6 release from U373 cells under serum-free
conditions, and this effect was enhanced by the presence of serum or
sCD14 (Fig. 4B). Hence, despite their different responses to
LPS in the absence of serum accessory proteins, both CHO cells and U373
cells responded to ER-112022 in the absence of serum, and the response
was enhanced by serum or sCD14. Similar results were obtained with the
endothelial cell line HMEC-1 (data not shown). RAW cells had a
different pattern of response. These cells responded to LPS in the
absence of serum, but serum enhanced the TNF release induced by LPS
(Fig. 4C). However, the ability of ER-112022 to activate
these monocyte/macrophage cells was not affected by serum (Fig.
4D). Hence, serum/sCD14 may have different effects on
ER-112022-mediated activation of nonmyeloid and myeloid cells. Our
results indicate that in serum-free medium, low doses of ER-112022
activate cells such as CHO-K1 and U373 that do not normally respond to
LPS under these conditions. When serum is added to U373 cells, the
potency of LPS and ER-112022 is comparable. Finally, upon stimulation
of RAW and CHO/CD14 cells in the presence of serum, ER-112022 is
considerably less potent than LPS on a mass basis in inducing cellular
activation.
The LPS Antagonists E5564 and E5531 Inhibit ER-112022 Activation of
Cells--
To determine whether LPS and ER-112022 interact with a
common recognition element in cells, a pharmacological strategy was applied, using the synthetic LPS antagonists E5564 (41) and E5531 (40)
(Fig. 1). This type of drug is thought to block interactions between
LPS and TLR4 (12, 23). As shown in Fig.
5A, E5564 inhibited ER-112022
but not TNF or IL-1 ER-112022 Activates HEK293 Cells That Have Been Co- transfected
with TLR4 Plus MD-2--
To test directly if the LPS-mimetic effects
of ER-112022 were due to its interaction with the LPS receptor, we
transiently transfected TLR4 and MD-2 into LPS nonresponder HEK293
cells. These cells do not express either TLR4 or MD-2 as assessed by reverse transcriptase-polymerase chain reaction (data not shown and
Ref. (14)). Transfectants were subsequently stimulated with ER-112022
or LPS. Neither compound activated HEK293 cells in the absence of gene
transfection or activated cells transfected with MD-2 alone (Fig.
6). In contrast, both ER-112022 and LPS
activated the NF- ER-112022 Activates Primary Monocytic Cells through TLR4--
To
investigate the role of TLRs in ER-112022 activation of monocytes, we
exposed freshly isolated adherent human monocytes to ER-112022, in the
presence or absence of antibodies to TLR2 or TLR4, and we measured the
release of TNF. Treatment with ER-112022 induced release of TNF,
although the efficacy was substantially less than that of LPS (Fig.
7A). As one might have
predicted from the cellular transfection data, the TLR4 antibody HTA125
completely blocked the TNF-inducing ability of ER-112022 and most of
the cellular activation induced by LPS (Fig. 7A). In
contrast, the TLR2 antibody TL2.1 did not have any effect on the
ER-112022 activation of primary monocytes, although TL2.1 inhibited
~70% of the TNF release mediated by the synthetic lipohexapeptide
47L, which we have previously demonstrated to be a ligand for TLR2
(21). The activation induced by 47L was not affected by the TLR4
antibody. Thus, ER-112022 and 47L appear to mediate TNF-inducing
ability in monocytes through two different receptors. By using
synthetic chemistry, it may be possible to determine precisely the
molecular structures necessary to activate either TLR2 or TLR4.
C3H/HeJ mice are hyporesponsive to LPS due to a dominant negative
mutation in tlr4 gene that results in a proline to
histidine substitution at position 712 (11, 18, 19). We exposed
peritoneal macrophages from C3H/HeJ and the LPS-responsive C3H/HeN mice
to ER-112022 and LPS. Both of these compounds induced TNF release from
the HeN macrophages; however, the HeJ macrophages did not respond to
LPS or ER-112022 (Fig. 7B). The lipopeptide 47L activated macrophages from the two mouse strains equally (data not shown).
The signaling events leading to the activation of cells by LPS are
not fully understood, although recent reports strongly implicate TLR4
and MD-2 as CD14-associated signaling components of an LPS receptor
complex. CD14 is the best characterized portion of the LPS receptor,
having been shown to actually bind LPS. However, multiple lines of
evidence suggest that the GPI-anchored CD14 is not an actual signal
transducer and that it does not account for the specificity of
biological responses to endotoxins. When dissecting the mechanisms of
signaling in the LPS receptor system, the use of both agonists as well
as antagonists may help increase the understanding of the complex
interactions between ligands and receptor molecules. Whereas neither
pharmacological nor molecular genetic approaches can replace
biophysical and biochemical analysis of receptor function, both
approaches can add substantially to our understanding of receptor mechanisms.
In this study, we describe ER-112022, a unique new acyclic and
symmetrical LPS mimetic. ER-112022 activates cells through the
LPS-signaling pathway via TLR4 and MD-2 but does not require membrane
or soluble CD14. This observation suggests that the role of CD14 in LPS
signaling may be limited to the simple transfer of LPS to TLRs and may
not involve the formation of a ternary complex involving TLR4/MD-2 and
CD14. The symmetrical organization of ER-112022 further suggests that
compounds of the correct size, shape, and hydrophobicity may cross-link
transmembrane signaling molecules, perhaps resulting in the formation
of a TLR4 homodimer, and thus the initiation of a proinflammatory signal.
A great deal of effort has been focused on the role of LPS receptor
cofactors present as soluble molecules in blood that augment the
potency of LPS in vitro and to a lesser (but important)
extent in vivo (51, 52). The data presented in this
work suggest a series of events that begin with LPS binding to
serum LPS-enhancing proteins that alter the conformation of the LPS
molecule itself. Transfer of LPS from soluble mediators like LBP and
sCD14 to membrane CD14, as well as transfer of LPS from membrane CD14
to TLR4, presumably occurs because of slight alterations in LPS
conformation that facilitate Toll receptor binding. ER-112022 seems to
have a structural conformation that enables it to bypass all of these
LPS-facilitating proteins in binding to TLRs and thus to interact
directly with the LPS signal transducer. The structural basis of this
remarkable physical property, when elucidated, should shed light on the
nature of the microenvironment of the putative LPS-binding pocket on TLR4.
The essential components necessary for activation of the LPS-signaling
pathway are present in both nonmyeloid and myeloid cells, and the data
to date suggest that in vitro work involving transfection of
nonmyeloid cells with genes encoding LPS receptors can be highly
informative about the nature of phagocyte LPS receptors. The unique
structure of the novel acyclic LPS-like agonist ER-112022 allows the
compound to interact directly with this pathway in a CD14-independent
manner. ER-112022 and derivatives may directly bind to the TLR4-MD-2
complex. Because of its defined synthetic basis, as well as its
independence from CD14 and LBP, ER-112022 should be useful for
simplifying both the binding and co-crystallization studies with TLR4
that will be necessary for identifying the region or regions of TLR4
that directly contact endotoxin. Hence, these studies may help to
design specific drugs for endotoxin-related diseases, including
Gram-negative bacterial septic shock.
B in HEK293 cells transfected
with TLR4/MD-2. Finally, tumor necrosis factor release from primary
human monocytes exposed to ER-112022 was blocked by TLR4 antibodies but
not by TLR2 antibodies. Our results suggest that ER-112022 and the
family of lipid A-like LPS antagonists can functionally associate with
TLR4 in the absence of CD14. Synthetic molecules like ER-112022 may
prove to be valuable tools to characterize elements in the LPS receptor
complex, as well as to activate or inhibit the TLR4 signaling pathway
for therapeutic purposes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 integrin family members CD11/CD18 (5). However,
neither CD14, which does not have a trans-membrane domain (6-8), nor
CD11/CD18 (5) appear to transduce a signal directly. Members of the
family of Toll-like receptors (TLR), especially TLR4, have recently
been suggested as LPS signal transducers (9-12). In addition, a small
protein, known as MD-2, enhances the ability of LPS to activate cells
via TLR4 (13) and appears to be a part of a TLR4 complex (14). The Toll
family member RP105 may also associate with TLR4 and mediate LPS
responsiveness in B cells (15). In addition to the membrane-bound
receptors, blood contains several factors, including soluble CD14
(sCD14) (16) and LPS-binding protein (LBP) (17), that are important
modulators of LPS activity.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. Ciprofloxacin was a gift from Miles;
penicillin/streptomycin was obtained from Sigma; hygromycin B was from
Calbiochem; and geneticin (G418) from Life Technologies, Inc. LPS
(Re595 LPS from Salmonella minnesota) was a gift from Drs.
N. Qureshi and K. Takayama (University of Wisconsin, Madison, WI);
alternatively, smooth LPS from S. minnesota or
Escherichia coli (from Sigma or List Biological Labs,
Campbell, CA) was used. Murine recombinant tumor necrosis factor-
(TNF) and human interleukin (IL)-1
were from Genzyme (Cambridge,
MA). Phorbol myristate acetate (PMA) was from Sigma. Recombinant human
sCD14 and LBP were gifts from H. Lichenstein (Amgen, Thousand Oaks,
CA); alternatively, sCD14 was purified by affinity chromatography as
described (12). The 47L synthetic lipohexapeptide based upon the
N-terminal structure of the 47-kDa major membrane lipoprotein from
Treponema pallidum was a gift from T. Sellati and J. D. Radolf, University of Connecticut. Antibodies (FITC anti-CD25/Tac and
isotype controls) for flow cytometry (FACS) experiments were purchased
from Becton Dickinson. Antibodies for inhibition experiments were
purified from hybridoma supernatant on Sepharose goat anti-mouse IgG as
described by the manufacturer (Zymed Laboratories
Inc., San Francisco, CA) and were stored as frozen stock
solutions in sterile PBS.
B reporter cell line CHO/ELAM.Tac was engineered by stable
co-transfection of the plasmid pCEP4 (Invitrogen, Carlsbad, CA) and the
NF-
B reporter plasmid pELAM.Tac (45), using calcium phosphate
precipitation as described previously (21). A clonal reporter cell line
was isolated from nonclonal "bulk" transfectants that survived
hygromycin selection by stimulating these cells overnight with
ER-112022 (300 ng/ml). Cells were subsequently positively selected for
a functional reporter transgene by cell sorting for the induced surface
expression of Tac (CD25) expression (Epics Elite cell sorter, Coulter,
Hialeah, FL) by staining the stimulated transfectants with anti-Tac
monoclonal antibody (Becton Dickinson). After expansion of the
enriched cells, the sort was repeated. Finally, to minimize the
background expression of the transgene, unstimulated cells were sorted
once for low CD25 expression. A clonal cell line (designated clone EL1)
was obtained by limiting dilution.
B-dependent luciferase reporter pELAM.luc (12, 45) and 25 ng of Renilla-luc (Promega, Madison, WI). After
18 h in DMEM, 1% FCS, the cells were washed and exposed to medium alone, sCD14, E. coli LPS, or ER-112022 plus sCD14 for
6 h. Cells were harvested in lysis buffer and analyzed for
luciferase activity using reagents from Promega (Dual Luciferase
Reporter System), by a Wallach 1450 MicroBetaTrilux counter. Firefly
luciferase activity in samples was normalized for transfection
efficiency by specific Renilla luciferase activity per the
manufacturer's instructions.
B Activation in CHO
Reporter Cell Lines--
Cells were stimulated for 18-22 h as
indicated, washed once with PBS, and harvested with trypsin-EDTA
(TLR2, TLR4, CD25, and CD14 are all resistant to the effects of
trypsin (data not shown)). All subsequent steps were performed on
melting ice. A cell pellet was resuspended in PBS, 1% FCS, and the
cells were labeled with a 1:30 dilution of the FITC-labeled anti-CD25
or isotype control antibodies (total labeling volume of 30 µl) for 30 min. Then, the cells were washed once with 2 ml of PBS/FCS and
resuspended in 0.3 ml of PBS/FCS containing 1 µg/ml propidium iodine
(Sigma) to identify dead cells. The cells were analyzed using a FACScan microfluorimeter (Becton Dickinson) as described previously (48).
B Electrophoretic
Mobility Shift Assay (EMSA)--
Preparation of nuclear extracts and
NF-
B EMSA were performed as described earlier (41). The binding
pattern of nuclear proteins to the 32P-labeled NF-
B
consensus oligonucleotide was visualized by separation on a 4%
nondenaturating polyacrylamide (Protogel, National Diagnostics, Atlanta, GA) gel, and dried gels were exposed to Biomax MS x-ray film
(Eastman Kodak Co.). In some experiments, a 100-fold excess of
unlabeled probe was used for specific blocking of NF-
B binding to
the radiolabeled oligonucleotide.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxo-amide chain linked closer to the
phosphodiester. Thus, the compound has three unique features compared
with naturally occurring lipid A from E. coli (Fig. 1).
First, ER-112022 is devoid of a cyclic carbohydrate backbone. Second,
the phosphates are phosphodiesters incorporated within the confines of
the structural backbone, unlike the phosphoesters on E. coli
lipid A, and finally, the structure is symmetrical (Fig. 1).
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Fig. 1.
Structure of compounds ER-112022, E5564,
E5531, and E. coli lipid A. The structure of the
LPS agonist ER-112022 (empirical formula of the disodium salt:
C88H162N4Na2O20P2,
Mr 1704.19) is compared with the E. coli lipid A and the LPS antagonists E5564 and E5531. Note that
ER-112022 does not contain the diglucosamine backbone found in lipid A,
E5564, and E5531.
B by EMSA. ER-112022, LPS, and TNF stimulated
the nuclear translocation of NF-
B in CHO/CD14, both in the absence
and presence of 1% human serum (Fig. 2).
This LBP-independent response is presumably due to the extremely high
levels of CD14 expression in CHO/CD14 (to observe serum or
LBP-dependent LPS stimulation of CHO/CD14, it is necessary
to test these cells with pg/ml concentrations of LPS, data not shown).
Unlike many cells that do not express membrane CD14, mock-transfected
CHO/Neo (or wild-type CHO-K1) cells are not enabled to respond to LPS
by the presence of soluble CD14 (Fig. 2). The basis of this resistance
to LPS stimulation is not known but is presumed to be due to the
failure of LPS to interact with TLRs expressed on the surface of CHO
cells. Surprisingly, ER-112022 was able to stimulate control CHO/Neo
cells under serum-free conditions (Fig. 2), although the cells remained
refractory to the effects of LPS under identical conditions. Extensive
washing of the cells did not alter the CD14-independent effect of
ER-112022 on NF-
B translocation in CHO/Neo cells. However, when the
CHO/Neo cells were activated in the presence of 1% human serum, which contains both sCD14 and LBP, the potency of ER-112022 was increased (Fig. 2). Thus, the effect of ER-112022 is markedly different from LPS,
which fails to activate CHO cells unless they are engineered to express
LPS-binding proteins, such as membrane-anchored CD14 (44), CD11/CD18
(5, 49), or GPI chimers of LBP or BPI (50), even in the presence
of soluble LBP and sCD14.
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Fig. 2.
ER-112022 activates CHO cells in the absence
of CD14 expression. CHO/Neo and CHO/CD14 cells were plated at a
density of 5 × 105/well in 6-well dishes. The
following day, cells were washed three times with PBS and stimulated
with ER-112022, TNF, or ReLPS in Ex-Cell serum-free medium in
the absence or presence of 1% human serum. After 1 h, the induced
nuclear translocation of NF- B was assessed by EMSA as described
under "Experimental Procedures." 0 means unstimulated
cells. No extr. represents 32P-labeled NF-
B
probe analyzed by EMSA in the absence of nuclear extracts.
CTR refers to extracts from cells stimulated with 100 ng/ml
ER-112022 that were analyzed in the presence of a 100-fold excess of
unlabeled probe. All concentrations given are in nanograms of stimulus
per ml. Shown is one experiment out of four.
B
reporter cells were exposed to ER-112022 in the absence or presence of recombinant sCD14 and recombinant LBP. The CHO reporter cell line responded to ER-112022, TNF, and IL-1
under serum-free conditions by
up-regulating membrane CD25 (Fig.
3A). Although CD14 was not necessary for a robust response to ER-112022, the presence of sCD14
enhanced ER-112022-induced NF-
B activation (Fig. 3B),
whereas recombinant LBP had no significant effect (data not shown). No synergistic effects were observed when both sCD14 and LBP were added
together with ER-112022 (not shown). Similar results were obtained when
NF-
B nuclear translocation in CHO-K1 and CHO/Neo was evaluated by
EMSA (not shown). The reporter cell line was hyporesponsive to LPS both
in the absence or in the presence of serum components (not shown), as
previously reported (21).
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Fig. 3.
Soluble CD14 enhances the ER-112022
stimulatory effect toward CHO reporter cells, whereas LBP does
not. CHO/Elam.Tac cells express CD25 as a consequence of NF- B
nuclear translocation. The reporter cells were plated at a density of
106 cells/well in 6-well dishes. The day after, they were
stimulated in serum-free medium with ER-112022 (100 ng/ml), TNF (10 ng/ml), or IL-1
(5 ng/ml) (A), or ER-112022 (10 ng/ml)
(B) in the presence or absence of 100 ng/ml of recombinant
sCD14. After 18 h, the cells were harvested, labeled with
FITC-CD25 antibody, and subjected to flow cytometric analysis. Given is
relative cell number on the y axis, and fluorescence on the
x axis. 0 means unstimulated cells. Adding
recombinant LBP (100 ng/ml) did not affect the stimulatory ability of
ER-112022, and sCD14 itself or LPS did not activate the cells. Shown is
one experiment out of three performed.
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Fig. 4.
ER-112022 induces cytokine release from U373
and RAW264.7 cells in a serum-independent but serum-enhancing
manner. A and B, U373 cells were plated at a
density of 2 × 104 cells/well in 96-well dishes. The
following day, cells were washed three times with Hanks' balanced salt
solution and stimulated with E. coli LPS in serum-free DMEM
with (open circles) or without (closed circles)
1% human serum or sCD14 (triangles, 150 ng/ml)
(A). Cells were stimulated with increasing amounts of
ER-112022 in serum-free DMEM (closed circles) or DMEM
containing 1% human serum (triangles) or sCD14 (open
circles, 150 ng/ml) (B). After 16-18 h of incubation,
supernatant was harvested and assessed for IL-6 concentration by ELISA.
Medium containing human serum or sCD14, or LPS in the absence of serum
components, did not induce IL-6 release. C and D,
RAW cells were plated in 96-well dishes at a concentration of 2 × 105 cells/well. The day after, the cells were washed three
times with serum-free DMEM and stimulated with E. coli LPS
(C) or ER-112022 (D) in DMEM in the absence
(closed triangles) or presence (open triangles)
of 1% FCS. Three hours later, the supernatant was harvested and
analyzed for TNF content by ELISA. Shown are the mean of triplicate
determinations ± S.D.
(data not shown) induction of NF-
B nuclear
translocation in CHO-K1 cells, even in the complete absence of serum
proteins. Similarly, the LPS antagonist E5531 potently inhibited
induced IL-6 release from U373 (Fig. 5B) and TNF release
from RAW cells (Fig. 5C) in a dose-dependent
manner. The data from these experiments support the following two
conclusions. 1) ER-112022 stimulates cells through the LPS-signaling
pathway, and 2) the LPS inhibitors E5564 and E5931 interact with the
LPS signal transducer in the absence of membrane or soluble CD14.
View larger version (18K):
[in a new window]
Fig. 5.
The LPS antagonists E5564 and E5531 inhibit
ER-112022-induced cellular activation, even under serum-free
conditions. A, CHO-K1 cells were plated as indicated in
Fig. 2 and exposed to ER-112022 with increasing amounts of the LPS
inhibitor E5564 in Ex-Cell serum-free medium, in the absence or in
presence of 100 ng/ml sCD14. After 1 h, nuclear extracts were
isolated, and nuclear translocation of NF- B was assayed by EMSA.
B, U373 cells were plated as given in Fig. 4, washed, and
exposed to ER-112022 (600 ng/ml) and increasing amounts of the LPS
inhibitor E5531 in serum-free DMEM (open triangles) or DMEM
containing sCD14 (circles, 150 ng/ml). After 16-18 h,
supernatant was harvested and analyzed in IL-6 ELISA. C, RAW
cells were plated as indicated in Fig. 4, washed, and exposed to
ER-112022 (600 ng/ml) in DMEM in the absence (closed
triangles) or presence (open triangles) of 1% FCS.
After 3 h, supernatant was harvested and subjected to TNF ELISA.
Given is triplicate determinations ± S.D. One representative
experiment out of three performed is shown.
B reporter in cells that co-expressed TLR4 and MD-2
(Fig. 6).
View larger version (30K):
[in a new window]
Fig. 6.
ER-112022 activates HEK293 cells transfected
with TLR4 and MD-2. HEK293 cells (3 × 105/well
in 24-well dishes) were allowed to adhere overnight. The cells were
transfected with the following plasmids: empty vector alone, MD-2, or a
combination of TLR4 and MD-2, together with the luciferase reporter
pELAM.luc and Renilla luciferase plasmid. After 18 h
the cells were exposed to medium alone, sCD14 (150 ng/ml), E. coli LPS (100 ng/ml) + sCD14 (150 ng/ml), or ER-112022 (1 µg/ml) + sCD14 (150 ng/ml). Six hours later, the cells were lysed and
subjected to analysis for luciferase activity. Given is relative
firefly luciferase activity after normalization by Renilla
luciferase activity.
View larger version (13K):
[in a new window]
Fig. 7.
ER-112022 activates primary cells via
TLR4. A, primary human adherent monocytes were isolated
in 24-well dishes as described under "Experimental Procedures." The
cells were preincubated for 20 min at room temperature in the absence
(triangles) or presence of 10 µg/ml of control antibody
6H8 (×), the TLR2 antibody TL2.1 (circles), or the TLR4
antibody HTA125 (squares), followed by exposure to
increasing amounts (0.03-1 µg/ml) of ER-112022, S. minnesota smooth LPS (10 ng/ml), PMA (1 ng/ml), or T. pallidum synthetic lipohexapeptide 47L (1 µg/ml) in serum-free
RPMI medium. After 8 h, the supernatant was harvested and analyzed
for TNF contents by bioassay. Results are shown as mean of duplicate
determinations ± S.D. of one experiment out of two performed.
B, peritoneal macrophages from TLR4 mutant C3H/HeJ
(squares) and control C3H/HeN (triangles) mice
were collected by lavage after thioglycollate injection. Cells (5 × 105 cells/well in 24-well dishes) were allowed to adhere
for 2 h; nonadherent cells were removed by washing, and cells were
stimulated with increasing amounts (0.1-1 µg/ml) of ER-112022 or
E. coli LPS (10 ng/ml) for 8 h in RPMI, 10% FCS.
Cell-free supernatant was harvested, and the TNF content was determined
by bioassay. Shown is the mean of triplicate determinations ± S.D. in one experiment out of two performed.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Robin R. Ingalls, Holger Heine, Atsutoshi Yoshimura, and Brian Monks for helpful discussions and comments and Unni Nonstad, Hua Yang, and Donna W. Young for technical assistance.
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FOOTNOTES |
---|
* This work was supported by the Norwegian Cancer Society, The Research Council of Norway (to E. L. and T. E.), and National Institutes of Health Grants GM54060, DK50305, and AI38515 (to D. T. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The 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, Norwegian University of Science and Technology, University Medical Center, 7489 Trondheim, Norway. Tel.: 47 73 55 02 91; Fax: 47 73 59 88 01; E-mail: egil.lien@medisin.ntnu.no.
Published, JBC Papers in Press, October 13, 2000, DOI 10.1074/jbc.M009040200
2 B. Naume and T. Espevik, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
LPS, lipopolysaccharide;
TLR, toll-like receptor;
LBP, LPS-binding protein;
sCD14, soluble CD14;
GPI, glycosylphosphatidylinositol;
CHO, Chinese
hamster ovary;
EMSA, electrophoretic mobility shift assay;
NF-B, nuclear factor
-B;
TNF, tumor necrosis factor
;
IL-1
, interleukin-1
;
PMA, phorbol myristate acetate;
FACS, fluorescence-activated cell scan;
FITC, fluorescein isothiocyanate;
DMEM, Dulbecco's modified Eagle's medium;
ELISA, enzyme-linked
immunosorbent assay;
FCS, fetal calf serum;
PBS, phosphate-buffered
saline.
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