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From the
Received for publication, February 11, 2002, and in revised form, June 18, 2002
The alginate capsule produced by the human
pathogen Pseudomonas aeruginosa is composed mainly of
mannuronic acid polymers (poly-M) that have immunostimulating
properties. Poly-M shares with lipopolysaccharide the ability to
stimulate cytokine production from human monocytes in a
CD14-dependent manner. In the present study we examined the
role of Toll-like receptor (TLR) 2 and TLR4 in responses to poly-M.
Blocking antibodies to TLR2 and TLR4 partly inhibited tumor necrosis
factor production induced by poly-M in human monocytes, and further
inhibition was obtained by combining the antibodies. By transiently
transfecting HEK293 cells, we found that membrane CD14 together with
either TLR2 or TLR4/MD-2 could mediate activation by poly-M.
Transfection of HEK293 cells with TLR2 and fluorescently labeled TLR4
followed by co-patching of TLR2 with an antibody revealed no
association of these molecules on the plasma membrane. However,
macrophages from the Tlr4 mutant C3H/HeJ mice and TLR4
knockout mice were completely non-responsive to poly-M, whereas the
tumor necrosis factor release from TLR2 knockout macrophages was half
of that seen with wild type cells. Taken together the results suggest
that both TLR2 and TLR4 are involved in cell activation by poly-M and
that TLR4 may be required in primary murine macrophages.
Alginate is a family of linear co-polymers of 1-4-linked
Our group has previously shown that alginates are able to stimulate
monocytes to cytokine production (3). The potency increases with the
content of M residues and molecular size, and polymers isolated from
mucoid pseudomonads (poly-M, 92-96% M) are the most active (3).
However, attaching oligomeric M blocks of low activity to particles
enhances their TNF-inducing potency by 2-4 log units, suggesting that optimal immunostimulating activity is determined from
the polymer conformation (4, 5). Given prophylactically, poly-M has
also been shown to protect mice from lethal Escherichia coli
infection2 and x-irradiation
(6) and to stimulate murine hematopoiesis (6).
Like lipopolysaccharide (LPS) from Gram-negative bacteria (7), poly-M
induces TNF production from human monocytes by binding to the surface
receptor CD14 (8), and the response is enhanced by serum as a source of
LPS-binding protein and soluble CD14 (5, 7, 9, 10). CD14 lacks an
intracellular part (11) and probably mediates cell activation by
interaction with other signal transducing molecules. Ten human
Toll-like receptors (TLRs) have been cloned, and some of them have been
shown to be microbial signal transducers (for review, see Ref. 12). The
LPS hyporesponsiveness of C3H/HeJ and C57BL/10ScCr mice is due to a
dominant negative point mutation and recessive null mutation,
respectively, in Tlr4 (13). Furthermore, TLR4 knockout
(TLR4 Reagents--
Poly-M (~350 kDa, 92% M) was isolated from
mucoid P. aeruginosa, and the batch was the same as that
previously used (4, 5). The endotoxin content was 6 ng/mg as measured
by the Limulus amebocyte lysate assay (Chromogenix AB,
Mõlndal, Sweden). LPS O26:B6 from E. coli, Re595 LPS
from Salmonella minnesota and polymyxin B
were purchased from Sigma. Phenol-reextracted protein-free LPS from
E. coli K235 (25) was kindly provided by Dr. S. N. Vogel (Bethesda, MD). The synthetic lipohexapeptide 47L, based upon the
N-terminal structure of the 47-kDa lipoprotein from Treponema pallidum, was a kind gift from Drs. T. Sellati and J. D. Radolf (University of Connecticut). Synthetic tetraacyl lipid A
precursor known as lipid IVA (compound 406) was provided by Dr.
S. Kusumoto (Osaka University, Toyonaka, Japan; Ref. 26). Synthetic
macrophage-activating lipopeptide (MALP)-2 from mycoplasma was a kind
gift from both Dr. P. F. Mühlradt (Gesellschaft für
Biotechnologische Forschung, Braunschweig, Germany), generated as
previously described (24), and Dr. G. Rawadi (Institute Pasteur,
Paris, France). Heat-killed Listeria monocytogenes was
kindly provided by Dr. G. Teti (University of Messina, Italy; Ref.
19).
The TLR2 monoclonal antibody (mAb), TL2.1, was generated in our
laboratory (19, 32), and the TLR4 mAb, HTA125, was a generous gift from
Dr. K. Miyake (Saga Medical School, Saga, Japan; Ref. 16). TL2.1,
HTA125, the CD14 mAb, 3C10 (ATCC, Manassas, VA), and the mAb 6H8
recognizing a widely distributed 180-kDa
glycoprotein3 were purified
from supernatants of the respective hybridoma cell lines on Sepharose
goat anti-mouse IgG as described by the manufacturer (Zymed
Laboratories Inc., San Francisco, CA). Recombinant human TNF
(specific activity, 7.6 × 107 units/mg) was supplied
by Genentech Inc. (South San Francisco, CA), and recombinant human IL-6
(specific activity, >1 × 108 units/mg) was purchased
from Genzyme Pharmaceuticals (Cambridge, MA).
Stimulation and Culture Conditions of Peripheral Blood
Mononuclear Cells and Cell Lines--
Peripheral blood
mononuclear cells were isolated from human A+ buffy coats (The
Bloodbank, RiT, Trondheim, Norway) by gradient centrifugation with
Lymphoprep as described by the manufacturer (Amersham Biosciences).
Adherent human monocytes (1-2 × 105 monocytes/well
in 24-well dishes) were washed three times in Hanks' balanced salt
solution (Invitrogen) and pretreated with a 10 µg/ml
concentration of the indicated mAbs or with 1 µg/ml synthetic lipid
IVA (compound 406) for 30 min at room temperature in serum-free RPMI
1640 medium (Invitrogen) with 0.01% L-glutamine, 40 µg/ml gentamycin (referred to as RPMI hereafter) prior to addition of
stimuli. Stimulation proceeded for 8 h at 37 °C before
supernatants were collected and stored at
Mock-transfected (neomycin) and CD14-transfected U373
astrocytoma cells were a kind gift from Dr. R. Thieringer (Merck
Research Laboratories). U373/neo and U373/CD14 cells were adhered
(5 × 104 cells/well in 24-well dishes) for 5 h
in RPMI and 1% heat-inactivated FCS (HyClone, Logan, UT) at 37 °C
and 5% CO2. Subsequently the cells were incubated
overnight with different stimuli in RPMI, 1% pooled human A+
serum. Supernatants were collected and stored at
Human embryonic kidney (HEK) 293 cells (ATCC) were grown in Dulbecco's
modified Eagle's medium (Invitrogen) with 0.01%
L-glutamine, 40 µg/ml gentamycin, (referred to as DMEM)
and 10% heat-inactivated FCS (HyClone) at 37 °C and 8%
CO2.
Transient Transfection and Luciferase Assay--
Transient
transfection was performed using PolyFect® Transfection
Reagent according to the manufacturer's procedures (Qiagen, Valencia,
CA). Cells were plated at a cell density of 2 × 104
cells/well in 48-well dishes and incubated for 24-48 h. Cells were
washed once in phosphate-buffered saline and transfected in 200 µl of
medium with 0.12 µg of the reporter plasmid pELAM-luc (29) and
0.12-0.15 µg each of pcDNA3-CD14 (HEK293 cells only), pRK7-TLR2
(a gift from Dr. C. Kirschning, Technical University of Munich,
Germany), pcDNA3-TLR4 (a gift from Drs. R. Medzhitov and C. Janeway, New Haven, CT), and/or pEFBOS-MD-2 (a gift from Dr. K. Miyake). pcDNA3 was used to adjust the amount of plasmid to a total
of 0.6 µg. All plasmids were isolated using EndoFree plasmid kits
(Qiagen). The next day cells were activated with poly-M (100 µg/ml),
LPS K235 (0.1 µg/ml), or MALP-2 (10 nM) for 8-14 h at
37 °C. According to the manufacturer's protocol (Promega, Madison,
WI), cytoplasmic extracts were prepared, and luciferase activity was
measured by a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA).
Results from duplicate wells are given as -fold induction of
mean luciferase light units with reference to unstimulated cells
transfected with the reporter plasmid plus MD-2.
Fluorescent TLR4 Constructs and Immunofluorescence
Microscopy--
PCR of TLR4 was performed on pcDNA3-TLR4. The PCR
fragments were digested with BamHI and XhoI and
cloned into pcDNA3 with YFP already inserted. The fluorescent
pcDNA3 vectors were kindly provided by F. Chan and M. Lenardo
(National Institutes of Health, Bethesda, MD). The YFP-TLR4 construct
produced functional and fluorescent TLR4 protein with C-terminal tagged
YFP when transfected into HEK293 cells. The stable cell line of
YFP-TLR4 in HEK293 was made by calcium phosphate transfection. Bulk
populations of stably transfected cells were selected in 1 mg/ml G418
sulfate (BioWhittaker), and fluorescent cells were isolated using a
fluorescence-activated cells sorter (BD Vantage, BD PharMingen).
Several clonal cell lines were obtained by limiting dilution. The
fluorescent HEK293 cell lines were cultivated in DMEM, 10% FCS with
0.5 mg/ml G418 (Sigma). Two days prior to transient transfection, the
YFP-TLR4 cells (2 × 104 cells/well) were plated in
35-mm glass-bottom tissue culture dishes (MatTek Corp., Ashland, MA).
Cells were washed once in phosphate-buffered saline and transfected in
100 µl with 200 ng each of pcDNA3-TLR2, pcDNA3-CD14,
and pEFBOS-MD-2 with EffecteneTM Transfection Reagent
according to the manufacturer's procedure (Qiagen). Patching
experiments were performed by adding 10 µg/ml TL2.1 or 10 µg/ml
3C10 in medium for 30 min at room temperature. The cells were then
washed twice before 10 µg/ml A647-labeled goat anti-mouse
immunoglobulins (Molecular Probes) were added. After 30-45 min at room
temperature, the cells were examined live with an inverted Zeiss LSM510
confocal microscope by use of 514 nm (YFP) and 633 nm (A647) lasers. To
minimize bleeding between the channels the images were produced by
performing single laser tracks.
Peritoneal Macrophages--
Peritoneal macrophages from C3H/HeN
and C3H/HeJ mice (Harlan Ltd., Oxon, UK) were collected by lavage 4 days after intraperitoneal injections of 3 ml of 3% thioglycollate
(Difco, Detroit, MI). Macrophages were adhered (5 × 105 cells/well in 24-well dishes) for 2 h, washed, and
incubated with stimuli in RPMI, 10% FCS for 8 h at 37 °C
before supernatants were collected and stored at
Generation of the TLR2-deficient and TLR4-deficient mice has been
described previously (14, 15). C57BL/6 wild type, TLR2 Poly-M Fails to Induce IL-6 Production from CD14-transfected U373
Cells--
The U373 astrocytoma cell line does not express CD14, but
the cells are responsive to LPS in the presence of serum as a source of
soluble CD14 (9). We have shown that although poly-M shares with LPS
the ability to stimulate monocyte TNF production in a CD14-dependent manner (8) and that serum or soluble CD14
enhances the response (10), serum or soluble CD14 cannot support
poly-M-induced IL-6 production from U373 cells (8). To examine whether
membrane CD14 is necessary for activation by poly-M, mock-transfected
and CD14-transfected U373 cells were compared for responses to LPS and
poly-M (Fig. 1A).
In the presence of 1% human serum, LPS induced
dose-dependent IL-6 production from U373/CD14 cells that
was 1-1.5 log units higher than that in non-transfected cells. Poly-M
failed to activate U373 cells even when the cells were transfected with
CD14 (Fig. 1A). U373 cells did express endogenous TLR4,
TLR6, and MD-2 but not TLR2 (reverse transcription-PCR data not
shown). We next transiently transfected U373/CD14 cells with an
ELAM-luciferase reporter plasmid together with combinations of TLR2,
MD-2, and TLR4 and analyzed NF- TLR2 and TLR4 Are Involved in Signaling TNF Production from Human
Monocytes in Response to Poly-M--
CD14 is involved in both LPS- and
poly-M-induced monocyte TNF production (7, 8), but LPS signal
transduction occurs through TLR4 in primary cells such as
monocytes/macrophages (13-15). Blocking mAbs were used to examine the
possible involvement of TLR2 and TLR4 in mediating poly-M-induced TNF
production from monocytes. As shown in Fig
2A, mAbs to CD14 (3C10), TLR2
(TL2.1), and TLR4 (HTA125) all inhibited TNF production induced by
poly-M, although with different efficiencies. From several experiments, inhibition by TL2.1 and HTA125 was 60-95% and 40-90%, respectively, but combinations of TL2.1 and HTA125 yielded greater inhibition than
either mAb alone (Fig. 2A and not shown). mAbs to CD14 and TLR4, but not to TLR2, blocked LPS-induced activation of monocytes (Fig. 2B), and the control mAb, 6H8, did not influence
stimulation by either LPS or poly-M (Fig. 2). Thus, poly-M and LPS
share the involvement of CD14 and TLR4, but in addition poly-M uses
TLR2 for inducing monocyte release of TNF.
The activity of LPS is dependent on the nature and number of acyl
chains in the lipid A part (30), and some lipid A analogs antagonize
LPS activation of human cells (30). In other species, some of these
molecules are agonists, and it has been demonstrated that TLR4 is
responsible for the species-specific recognition of lipid A structures
(31). As seen in Fig. 2C, synthetic lipid IVA (compound 406)
inhibited both LPS- and poly-M-induced TNF production, supporting the
results obtained with blocking mAbs that poly-M is recognized by TLR4.
The stimulation of monocytes by poly-M was not due to contaminating LPS
since polymyxin B, which binds to the lipid A part of LPS, inhibited
activation by LPS and not by poly-M (Fig. 2D).
Poly-M Can Use Either CD14/TLR2 or
CD14/TLR4/MD-2 to Activate HEK293 Cells--
We
next sought to delineate the minimum receptor requirement of
poly-M-induced cell activation. HEK293 cells lacking CD14, TLR2, TLR4,
and MD-2 (data not shown) were transiently transfected with
combinations of these receptors together with an ELAM-luciferase reporter plasmid and analyzed for NF-
HEK293 cells transfected with TLR2, TLR4, and/or MD-2 were unresponsive
to poly-M unless membrane CD14 was expressed, indicating that, in
contrast to LPS, soluble CD14 cannot substitute for the membrane-bound
form in cell activation by poly-M (data not shown). HEK293 cells
co-transfected with CD14 and TLR2 responded to poly-M with NF- TLR2 and TLR4 Are Not Preassociated on the Plasma Membrane of
HEK293 Cells--
Since poly-M was found to signal through both TLR2
and TLR4 with CD14 or CD14/MD-2 as co-receptors, respectively, we found it important to examine whether TLR2 and TLR4 co-localized on the
plasma membrane in live cells. To perform these studies we engineered a
TLR4 construct with YFP on the C terminus of the molecules. The
fluorescent TLR4 was functionally active when transfected into
HEK293 cells.4 TLR2 or
CD14 was transiently transfected into HEK293 cells stably expressing
YFP-TLR4 and patched with antibodies to TLR2 (TL2.1) and CD14 (3C10),
respectively. As seen in Fig. 4,
A-C, TLR4 did not accumulate in TLR2 patches, indicating
that TLR2 and TLR4 are not preassociated on the plasma membrane of
HEK293 cells. Incubation with poly-M to induce receptor complex
formation or co-transfecting CD14 and MD-2 together with TLR2 did not
change this pattern (data not shown). In contrast, patching of CD14
resulted in pronounced co-localization of TLR4 (Fig. 4,
D-F), suggesting a close association between these two
membrane molecules. Similar results were obtained when cells expressing
YFP-TLR6 were transfected with TLR2 and patched with TL2.1 (data not
shown).
Macrophages from Tlr4 Mutant C3H/HeJ and
TLR4
To directly assess the relative importance of TLR2 and TLR4 in
mediating activation by poly-M, we compared poly-M-induced TNF
production in macrophages from wild type, TLR4 Medzhitov and Janeway (33) have proposed that innate immune
cells express pattern recognition receptors that discriminate between
self and non-self by recognition of conserved pathogen-associated molecular patterns. Several of the pattern recognition receptors, like
CD14 (34) and the TLRs (12, 33), recognize microbial components with no
apparent structural similarity. Poly-M was the first CD14 ligand
different from LPS to be described (8). Here we show that poly-M is
also recognized by TLR2 and TLR4. The finding that poly-M could not
activate U373 cells implies that these cells lack a component(s)
different from CD14, TLR2, TLR4, and MD-2 that is needed for responses
to poly-M, but not to LPS, and that is expressed in
monocytes/macrophages and HEK293 cells. This result supports a model in
which specific host responses are mediated by combinations of molecules
rather than by single pattern recognition receptors.
TLR4 is shown to mediate species-specific recognition of lipid A
structural analogs (31), and we found that synthetic tetraacyl lipid A,
also known as lipid IVA or compound 406, inhibited both LPS- and
poly-M-induced TNF production at low concentrations of antagonist. In a
previous report we found that the synthetic pentaacyl analog of
Rhodobacter capsulatus lipid A, B975, inhibited activation of human monocytes induced by LPS, but not by poly-M, in serum-free conditions (5). The reason for this discrepancy is not known but may be
related to differences in the ability to inhibit signaling through
TLR4, especially in the absence of serum (35).
Various investigators have pointed to the problem of purifying
bacterial components to homogeneity (36), and several data in the
present and previous works confirm that the results obtained with
poly-M are not due to contaminating LPS. First, the U373/CD14 cells
were sensitive to very low concentrations of LPS (less than 0.1 ng/ml,
not shown) but completely insensitive to poly-M. Second, polymyxin B,
which binds to lipid A and blocks LPS responses, did not affect
activation of human monocytes with poly-M (Fig. 1D and Ref.
3). Third, the TNF-inducing capabilities of high molecular
poly-M is greatly reduced by hydrolytic or enzymatic breakdown but
restored or even enhanced when the resultant low molecular chains are
attached to particles (4, 5). Finally, the lack of response to poly-M
in TLR4 The first sets of experiments with human monocytes indicated that CD14,
TLR2, and TLR4 are involved in mediating cell activation by poly-M. To
further address the relative contribution and importance of TLR2 and
TLR4, we used two other experimental systems: gain-of-function studies
in the human cell line HEK293 and loss-of-function studies in
TLR-defective murine macrophages. The results from HEK293 cells suggested that two separate signaling pathways exist for poly-M as
expression of either CD14/TLR2 or CD14/TLR4/MD-2 was sufficient for
NF- The immunomodulating properties of alginate are of commercial
importance as alginate is used in several biomedical applications (43),
and alginate capsules are currently being evaluated for use as
implantation material for hormone-producing cells (44, 45). Moreover,
carbohydrates have interesting potentials for treatment of various
infections and cancers (46). In addition to its cytokine-inducing
effect, antitumor activity has been demonstrated for algal high M
alginates (47). Furthermore, poly-M protects mice from lethal
x-irradiation and bacterial infections without imparting toxicity
(6).2 Thus, a possible exploitation of poly-M could be as a
general immunostimulator for protection against disease.
*
This study was carried out with financial support from the
Commission of the European Communities specific research, technological development, and demonstration (RTD) program "Quality of Life and
Management of Living Resources," Grant QLK2-2000-336,
HOSPATH; the Norwegian Cancer Society; and the Norwegian Research
Council.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. E-mail:
terje.espevik@medisin.ntnu.no.
Published, JBC Papers in Press, June 27, 2002, DOI 10.1074/jbc.M201366200
2
T. Espevik and G. Skjåk-Bræk, unpublished results.
3
T. Espevik and B. Naume, unpublished observation.
4
E. Latz, A. Visintin, E. Lien, K. Fitzgerald, B. G. Monks, E. Kurt-Jones, D. Golenbock, and T. Espevik, unpublished data.
The abbreviations used are:
M, D-mannuronic acid;
poly-M, mannuronic acid polymers;
TLR, Toll-like receptor;
LPS, lipopolysaccharide;
TNF, tumor necrosis
factor;
ELAM, endothelial leukocyte adhesion molecule;
MALP, macrophage-activating lipopeptide;
mAb, monoclonal antibody;
IL, interleukin;
FCS, fetal calf serum;
HEK, human embryonic kidney;
DMEM, Dulbecco's modified Eagle's medium;
YFP, yellow fluorescent
protein.
Involvement of Toll-like Receptor (TLR) 2 and TLR4 in Cell
Activation by Mannuronic Acid Polymers*
,,,,
,**
Institute of Cancer Research and Molecular
Biology and the Institute of Biotechnology, Norwegian University
of Science and Technology, 7489 Trondheim, Norway, the
§ Department of Medicine, Division of Infectious Diseases,
University of Massachusetts Medical School, Worcester, Massachusetts
01655, and the ¶ Department of Host Defense, Research Institute
for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-mannuronic acid
(M)1 and
-L-guluronic acid with highly variable composition and
sequential structure depending on the source from which it is isolated
(for review, see Ref. 1). In nature, alginate is found mainly as the
structure substance of marine brown seaweed but also as an exopolysaccharide produced by bacteria, like the opportunistic pathogen
Pseudomonas aeruginosa that colonizes patients with cystic fibrosis (2) and may cause severe infections in critically ill patients.
/) mice are non-responsive to LPS, whereas TLR2/ mice
respond normally (14, 15). In addition to TLR4, a soluble protein,
MD-2, associates with TLR4 and is needed for efficient LPS signaling
(16). Two uronic acid-containing polysaccharides are also found to
induce cell activation through TLR4, namely the Gram-positive
Micrococcus luteus teichuronic acids (17) and the fungal
Cryptococcus neoformans glucuronoxylomannan (18). TLR2
recognizes a range of different structures such as several
Gram-positive bacterial components (15, 19-21); yeast zymosan (21);
mycobacterial lipoarabinomannan (22); and lipoproteins and lipopeptides
from spirochetes, mycobacteria, and mycoplasma (20, 23, 24). The
present study was undertaken to examine whether TLR2 or TLR4 is
involved in signaling cytokine production induced by poly-M.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C until assayed for
TNF activity in the WEHI 164 clone 13 bioassay as described previously
(27). Results from one representative experiment are presented as
mean ± S.D. of triplicate TNF measurements.
20 °C until
assayed for IL-6 by the B9 cell proliferation assay (28). Results from
representative experiments are presented as mean ± S.D. of
triplicate IL-6 measurements.
20 °C until
assayed for TNF activity in the WEHI 164 clone 13 bioassay as described
previously (27). Results from one representative experiment are
presented as mean ± S.D. of triplicate TNF measurements.
/, and
TLR4/ mice were intraperitoneally injected with 2 ml of 4%
thioglycollate (Difco). Three days later, peritoneal exudate cells were
isolated from the peritoneal cavity by washing with ice-cold Hanks'
balanced salt solution. Cells were cultured for 2 h and washed
with Hanks' balanced salt solution to remove non-adherent cells.
Adherent monolayer cells (5 × 104 cells/well) were
used as peritoneal macrophages and cultured in RPMI, 10% FCS in the
presence of stimulants for 24 h. Supernatants were collected and
assayed for TNF by enzyme-linked immunosorbent assay according to the
manufacturer's instructions (Genzyme Pharmaceuticals). Results from
one representative experiment are presented as mean ± S.D. of
triplicate TNF measurements.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
U373 cells are non-responsive to poly-M.
A, mock-transfected or CD14-transfected U373 cells were
stimulated with the indicated concentrations (µg/ml) of E. coli O26:B6 LPS or poly-M, and supernatants were assayed for IL-6
bioactivity. Spontaneous IL-6 production was ~110 pg/ml. Results from
one representative experiment of three are shown (mean ± S.D. of
triplicate wells in the IL-6 assay). B, U373 cells stably
expressing CD14 (U373/CD14) were transiently transfected with an
ELAM-luciferase reporter plasmid together with the indicated constructs
and stimulated with poly-M (100 µg/ml), E. coli K235 LPS
(0.1 µg/ml), or MALP-2 (10 nM). Results are given as
-fold induction of mean luciferase light units with reference to
unstimulated cells transfected with MD-2. Results from one of two
similar experiments are shown. Unstim., unstimulated.
B activation by poly-M (Fig.
1B). Expression of TLR2 made U373/CD14 cells responsive to
the TLR2-TLR6 ligand MALP-2 (24), confirming a functional TLR2.
Similarly, overexpression of MD-2 and TLR4 resulted in increased
NF-B activation induced by LPS. However, none of the receptor
combinations were sufficient for poly-M to induce NF-B translocation
in U373 cells. Thus, the results in Fig. 1B demonstrate that
the inability of poly-M to activate U373 cells was not due to the
absence of TLR2 or low level expression of TLR4 or MD-2, suggesting
that U373 cells lack other components needed for responses to poly-M.
Altogether these results demonstrate that LPS and poly-M have different
requirements for inducing cell activation.
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Fig. 2.
Involvement of CD14, TLR2, and TLR4 in
activation of human monocytes. Adherent human monocytes were
untreated (Medium) or pretreated with a 10 µg/ml
concentration of mAbs to CD14 (3C10), TLR2
(TL2.1), or TLR4 (HTA125) or a control mAb
(6H8) before addition of poly-M (10 µg/ml) (A)
or E. coli O26:B6 LPS (0.1 µg/ml) (B) in
serum-free conditions. Results from one of four experiments are shown
as mean TNF ± S.D. of triplicate wells. In C, cells
were pretreated with 2 µg/ml HTA125 or 1 µg/ml synthetic lipid IVA
(406) prior to addition of poly-M (10 µg/ml) or E. coli O26:B6 LPS (0.1 µg/ml). Results, given as percentages of
the TNF level induced by poly-M (2.8 ± 0.15 ng/ml) or LPS
(12.8 ± 1.6 ng/ml) in the absence of pretreatment
(Medium), are from one representative experiment repeated
three times. In D, 5 µg/ml polymyxin B was added to the
cells prior to stimulation with poly-M (20 µg/ml) or E. coli O26:B6 LPS (0.1 µg/ml). Results are calculated as
percentages of TNF production stimulated by poly-M (11.0 ± 1.9 ng/ml) or LPS (117 ± 25 ng/ml) without polymyxin B. Shown are
data from one representative experiment repeated twice. The spontaneous
level of TNF was less than 100 pg/ml in all experiments.
B activation by poly-M (Fig. 3).
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Fig. 3.
Poly-M can use either CD14/TLR2 or
CD14/TLR4/MD-2 to activate HEK293 cells. HEK293 cells were
transiently transfected with an ELAM-luciferase reporter plasmid
together with the indicated constructs and stimulated with poly-M (100 µg/ml), E. coli K235 LPS (0.1 µg/ml), or MALP-2 (10 nM) for 8 h at 37 °C. Luciferase activity from
cytoplasmic extracts was measured, and results are given as -fold
induction of mean luciferase light units with reference to unstimulated
cells transfected with MD-2. One representative experiment of five is
shown. Unstim., unstimulated.
B
activation, and MD-2 was not required for signaling (Fig. 3). The same
pattern was observed for MALP-2. In contrast, expression of CD14 and
TLR4 was not sufficient to mediate poly-M responses, and additional
co-transfection with MD-2 was needed for poly-M to induce translocation
of NF-B (Fig. 3). Although we did not adjust for transfection
efficiency, no obvious improvement of poly-M-induced NF-B activation
was obtained by co-transfection with TLR2 and TLR4 ± MD-2. MD-2
was also required together with TLR4 to signal LPS activation in HEK293
cells. These results indicate that poly-M can use either CD14/TLR2 or
CD14/TLR4/MD-2 for signal transduction.
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Fig. 4.
Patching of TLR2 and CD14 in HEK293 cells
expressing YFP-TLR4. A and D, YFP-TLR4
distribution on the plasma membrane; B, TLR2 patched with
TL2.1; E, CD14 patched with 3C10; C and
F, overlaid images. As can be seen from the figure, the CD14
patches also contain TLR4 (F, arrows), whereas
patching of TLR2 did not recruit TLR4 (C,
arrows).
/ Mice Do Not Respond to Poly-M, whereas
TLR2/ Cells Are Partly Responsive--
The
importance of TLR4 in mediating LPS signaling was revealed by the
finding that the genetic defect in LPS-hyporesponsive C3H/HeJ mice is a
P712H missense mutation in Tlr4 (13). We next used
peritoneal macrophages isolated from C3H/HeJ mice and LPS-responsive C3H/HeN mice to examine the significance of TLR4 in signaling activation by poly-M. Macrophages from HeN mice responded to poly-M by
TNF production in a dose-dependent manner, whereas HeJ
macrophages were unresponsive even to 200 µg/ml poly-M (Fig.
5A, right). Similar results were obtained with LPS, although the HeN macrophages were far
more sensitive to LPS than to poly-M (Fig. 5A,
left). A TLR2 ligand, lipohexapeptide 47L from T. pallidum (20), induced comparable amounts of TNF production in HeN
and HeJ macrophages (Fig. 5A, middle). These
results confirm that HeJ mice express functional TLR2 and further
indicate that TLR4 may be necessary for induction of TNF production by
poly-M.
View larger version (29K):
[in a new window]
Fig. 5.
Induction of TNF production from mutant or
TLR2- or TLR4-deficient murine macrophages. A,
thioglycollate-elicited macrophages from C3H/HeN and C3H/HeJ
mice were incubated with E. coli K235 LPS, synthetic
lipohexapeptide from T. pallidum (47L), or poly-M
at the indicated concentrations (µg/ml) for 8 h in the presence
of 10% FCS before supernatants were harvested and assayed for TNF
bioactivity. The spontaneous TNF release was 40 pg/ml. Results from one
of three representative experiments are shown as mean ± S.D. of
triplicate wells in the TNF assay. B,
thioglycollate-elicited macrophages from wild type, TLR2 /, or
TLR4/ mice were incubated with the indicated concentrations of
poly-M (µg/ml), S. minnesota Re595 LPS (1 µg/ml), or
MALP-2 (0.3 ng/ml) for 24 h in the presence of 10% FCS before
supernatants were collected and assayed for TNF in enzyme-linked
immunosorbent assay. The spontaneous TNF level was undetectable, and
the experiment was repeated once with similar results.
/, and TLR2/ knockout mice. Poly-M triggered a dose-dependent TNF
release from wild type macrophages that was completely absent in
TLR4/ cells (Fig. 5B), confirming the results obtained
with HeJ macrophages (Fig. 5A). Macrophages from TLR2/
mice showed a reduced but significant response to poly-M. The TNF level
produced by TLR2/ cells was ~50% compared with that of wild type
cells (Fig. 5B). LPS-induced TNF production was comparable
from wild type and TLR2/ cells but absent in TLR4/ cells.
Moreover, MALP-2 activated macrophages from wild type and TLR4/
mice but not from TLR2/ mice, altogether confirming that the
function of TLR2 and TLR4 was normal in TLR4- and TLR2-deficient cells,
respectively. Thus, although TLR2 participates in poly-M-induced TNF
release, these results suggest that TLR4, and not TLR2, is required for
poly-M responses in primary murine macrophages.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ cells rule out possible contamination with lipoproteins or lipopeptides.
B translocation. TLR4 is proposed to form homodimers (21) that
together with MD-2 could mediate poly-M activation. As suggested by
Ozinsky et al. (37) TLR2 does not signal as homodimers but induces cytokine production in heteromeric complexes with TLR1 or TLR6
for instance (37-40). Co-patching experiments of YFP-TLR4-expressing HEK293 cells transfected with TLR2 or CD14 revealed that CD14, but not
TLR2, was preassociated with TLR4 on the plasma membrane. However, we
cannot exclude that TLR2 and TLR4 may interact in other cells, such
as primary macrophages. The results from TLR2- and
TLR4-deficient mouse peritoneal macrophages clearly indicated that
TLR4, and not TLR2, is required for cytokine induction by poly-M.
Nevertheless, the reduced TNF response in TLR2-deficient cells argues
for a role of TLR2, possibly together with TLR4. The reason for this
divergence is unclear but may be due to differences in cell systems and
experimental setup. We suggest that primary cells may better mirror the
responses that can occur in vivo. Common observations in all
cell systems, both primary cells and cell lines, lead us to the
conclusion that the induction of cell activation by poly-M involves
CD14, TLR2, and TLR4, and we can hypothesize a mechanism where TLR2 and
TLR4 both participate in mediating an optimal response to poly-M. In
addition to poly-M, glycolipids from Enterococcus hirae (41)
and human and chlamydial HSP60 (42) have also been reported to signal
through CD14, TLR2, and TLR4, but none of the studies addressed the
question of whether TLR2 and TLR4 worked together in mediating
signal transduction.
FOOTNOTES
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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K. Sau, S. S. Mambula, E. Latz, P. Henneke, D. T. Golenbock, and S. M. Levitz The Antifungal Drug Amphotericin B Promotes Inflammatory Cytokine Release by a Toll-like Receptor- and CD14-dependent Mechanism J. Biol. Chem., September 26, 2003; 278(39): 37561 - 37568. [Abstract] [Full Text] [PDF]
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