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*
The Pulmonary Center, Boston University School of Medicine, Boston, MA 02118; and
Infectious Disease Section, Boston Medical Center, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. The
central role played by NF-B in signal-transduction pathways
activated by cytokines, and in the regulation of cytokine genes
themselves, implicated mammalian Toll proteins in cellular responses
similar to those evoked by cytokines. Third, the intracellular domain
of Toll shares significant sequence similarity with the type I IL-1
receptor, the known mammalian Toll-like receptors, and the cytosolic
adapter protein MyD88 (4). Indeed, signaling via TLR4 has
been shown to require both MyD88 and the IL-1 receptor-associated
kinase (IRAK) (5). The shared use of a cytokine receptor
signaling pathway suggests that Toll, and its mammalian cousins, may
function as cytokine receptors. Indeed, Toll is a transmembrane
receptor protein with a known protein ligand in the fly (Spatzle).
Although mammalian ligands have not yet been reported, it has been
speculated that the five published members of the human TLR family
might recognize a family of cytokine-like ligands that participate in
host immune responses (6).
An unexpected feature of TLR biology comes from reports that these
proteins participate in intracellular signaling initiated by
Gram-negative bacterial LPS. CD14 is the major receptor responsible for
the effects of LPS on macrophages, monocytes, and neutrophils (reviewed
in Ref. 7). The role of CD14 in signaling has remained
unclear because it is a glycophosphatidylinositol-linked protein that
lacks transmembrane and intracellular domains. Several studies have
suggested that CD14 acts by associating with a distinct transmembrane
signal transducing protein. Recently published data support the
possibility that TLR proteins may serve this function. Two groups
independently reported that TLR2 could function as a signaling receptor
for LPS in the presence of CD14 (8, 9). These
investigators reported that HEK 293 (human 293 embryonic kidney) cells
stably transfected with TLR2 could respond to LPS in the presence of
CD14 and LPS binding protein, as judged by activation of a reporter
gene under the control of the NF-B-dependent endothelial
cell-leukocyte adhesion molecule-1 (ELAM-1) (E-selectin) promoter.
Deletion mutants of TLR2 lacking the 13 most C-terminal amino acids of
the intracellular domain failed to mediate LPS responsiveness in this
assay. Thus, TLR2 appears to mediate LPS-induced intracellular
signaling initiated by the binding of LPS to CD14. Most recently, it
has been shown that LPS induces the oligomerization of TLR2 and the
subsequent recruitment of the IRAK to the TLR2 complex
(10).
A different facet of this story was revealed by identification of the gene responsible for the LPS hyporesponsive phenotype of the C3H/HeJ mouse (11, 12, 13). Macrophages from this mouse are markedly resistant to activation by LPS, even though they express normal amounts of CD14 on their surface. Although the genetic defect in these mice was known to arise from a single locus (lpsd), the gene responsible for this defect remained elusive. Positional cloning and sequencing of the lpsd locus mapped the defect to the tlr4 gene. In C3H/HeJ mice, a single missense mutation within the tlr4 coding sequence was identified (P712H). Evidence that this mutation is responsible for the LPS hyporesponsive phenotype of the C3H/HeJ mouse comes from the finding that the C57BL/10ScCr LPS-nonresponsive mouse does not express TLR4. More recently, both macrophages and B cells from a TLR4 knockout mouse were shown to be LPS hyporesponsive (13). Lastly, it has been reported that TLR4 can confer CD14-dependent LPS responsiveness on HEK 293 cells (14), although LPS responsiveness also depends on the concomitant expression of an additional protein, MD-2 (15). Together, these findings implicate both TLR2 and TLR4 in LPS signal transduction.
Data from our laboratory and others suggest that distinct CD14 ligands possess different requirements for CD14-associated signal-transduction molecules. Savedra et al. (16) previously reported that Chinese hamster ovary (CHO) cells engineered to express human CD14 (CHO/CD14) could be activated by LPS, but not by a distinct CD14 ligand, mycobacterial lipoarabinomannan (LAM). This observation led us to hypothesize that different CD14 ligands might require distinct signal-transduction proteins to initiate intracellular signaling. In this study we tested the possibility that CD14-mediated cellular activation initiated by LAM and LPS requires distinct TLR proteins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CHO-K1 fibroblast, THP-1 human monocytic leukemia, and RAW264.7 murine macrophage cell lines were purchased from the American Type Culture Collection (Manassas, VA). THP-1 and RAW264.7 cells were maintained in DMEM culture medium (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT), 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (BioWhittaker). CHO cells were maintained in HAM F-12 culture medium (BioWhittaker) supplemented with 10% heat-inactivated FBS (HyClone), 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (BioWhittaker). Cells were cultured at 37°C in the presence of 5% CO2 in a humidified incubator. CHO/CD14, 3E10 (CHO/CD14/ELAM-CD25), 3E10/TLR2, and CHO/TLR2 were previously described (16, 17) and cultured as described above. Several clonal isolates of each cell line were examined, and identical results were obtained using each isolate. LPS levels in all medium components were <10 pg/ml final concentration as indicated by BioWhittaker or as measured by Limulus amebocyte lysate kit (BioWhittaker).
C3H/FeJ and C3H/HeJ mice (female, 5–6 wk old) were obtained from The
Jackson Laboratory (Bar Harbor, ME). All mice were used within 1 wk of
receipt. Mice were maintained in a laminar flow hood in cages fitted
with polyester filter hoods and fed standard lab food and acid water ad
libitum. Macrophages were elicited by i.p. injection of 2 ml sterile
fluid thioglycollate medium (3%) and subsequently harvested by
peritoneal lavage 4 days after injection. Adherent cells were cultured
at 37°C and 5% CO2 at a density of 
25
x 105 cells/ml in RPMI 1640 (BioWhittaker)
supplemented with 2% FCS, 2 mM glutamine, 30 mM HEPES, 0.4% sodium
bicarbonate, 100 IU/ml penicillin, and 100 µg/ml streptomycin as
described previously (18).
LPS (purified from Escherichia coli 055:B5) was purchased from Sigma (St. Louis, MO). LAM (purified from a rapidly growing avirulent mycobacterium) was provided by Dr. John Belisle (Colorado State University, Fort Collins, CO) under the provisions of National Institutes of Health Contract NO1-AI25147. Levels of contaminating LPS in the LAM preparations were determined using a quantitative Limulus lysate assay (BioWhittaker) and were less than 1 pg/ml final concentration in all experiments. We found that all LAM preparations were unable to activate the LPS-sensitive (and LAM-nonresponsive) CHO/CD14 cells, thus further demonstrating that the LAM preparations were not contaminated with LPS. Recombinant human IL-1ß was purchased from Genzyme (Cambridge, MA). FITC-labeled anti-human CD25 Abs were purchased from Becton Dickinson (San Jose, CA).
EMSA
Nuclear extracts were prepared essentially as described by Schreiber et al. (19). Approximately 1.0 x 107 CHO or RAW264.7 cells were washed and harvested by scraping in Ca2+- and Mg2+-free PBS (BioWhittaker). Cells were pelleted by centrifugation at 800 x g for 10 min at 4°C. Cell pellets were resuspended in 400 µl of a buffer containing 10 mM KCl, 10 mM Tris-HCl (pH 7.8), 5 mM MgCl2, 0.5 mM DTT, 0.3 M sucrose, 10 mM ß-glycerol phosphate, 0.1 mM EGTA, 1 mM PMSF, and 5 µg/ml each of aprotinin, leupeptin, chymostatin, and antipain and incubated on ice for 10 min. Subsequently, 25 µl of 10% Nonidet P-40 (Sigma) was added to each sample before vortex mixing. The nuclei were centrifuged for 1 min at 5000 x g to pellet the nuclei. Supernatants were collected and stored frozen for later use as cytosolic lysates. Nuclear pellets were resuspended in a nuclear extraction buffer containing 320 mM KCl, 10 mM Tris-HCl (pH 7.8), 5 mM MgCl2, 0.5 mM DTT, 10 mM ß-glycerol phosphate, 0.1 mM EGTA, 25% glycerol, 1 mM PMSF, and 5 µg/ml each of aprotinin, leupeptin, antipain, and chymostatin. Samples were extracted on ice for 15 min and then centrifuged at 16,000 x g for 10 min at 4°C. Protein concentration was determined using the Bio-Rad assay kit (Hercules, CA). All nuclear extracts were stored at -70°C and multiple freeze-thawing cycles were avoided.
A double-stranded oligonucleotide containing a single copy of the IL-2
receptor 
-chain NF-B (GGGGAATTCC) was used as an EMSA probe. DNA
probes were labeled with
[-32P]deoxynucleotide triphosphates
(DuPont/NEN, Boston, MA) using E. coli DNA polymerase Klenow
fragment (United States Biochemicals, Cleveland, OH) as recommended by
the manufacturer. Unincorporated nucleotides were removed using
Sephadex G-25 columns (5 Prime 
3 Prime, Boulder, CO). Nuclear
extracts (typically 3 µg) were incubated with radiolabeled probe DNA
(0.1 ng, typically 10,000 cpm) in the presence of 2 µg poly dI-dC
(Pharmacia, Piscataway, NJ), 1.0 mM EDTA, 10 mM Tris-HCl (pH 7.9), 25
mM glycerol, and 0.5 mM DTT in a final volume of 20 µl, as previously
described (19). Binding reactions were then incubated at
room temperature for 30 min. After incubation, a portion of each
binding reaction (typically 6 µl) was loaded onto 7% nondenaturing
low ionic strength polyacrylamide gel. The gels were then dried and
visualized by autoradiography.
Plasmids
The ELAM-luc reporter plasmid was generated by subcloning the
promoter of pUMS(ELAM)-Tac (17) into the promoterless pGL3
luciferase reporter plasmid (Promega, Madison, WI). The human TLR1,
TLR2, and TLR4 cDNAs cloned into the pFlag-CMV-1 mammalian expression
plasmid were gifts of Drs. Carsten Kirschning and Mike Rothe (Tularik,
South San Francisco, CA) and were previously described
(9). These gene products are expressed as fusion proteins
containing an N-terminal Flag epitope tag. A second TLR4 expression
plasmid that expresses native TLR4 (termed hToll) was a gift of Dr.
Charles Janeway (Yale University, New Haven, CT) and was previously
described (2). The TLR2
13 mutant expression plasmid
containing a Flag-TLR2 fusion cDNA that lacks the C-terminal 13 amino
acids of the TLR2 coding sequence (residues 771–784) was generated by
PCR. Plasmids were prepared using Qiagen (Chatsworth, CA) plasmid DNA
purification columns. DNA was eluted from the columns using LPS-free
buffers, and contaminating LPS levels were found to be less than 10
pg/ml. Furthermore, all plasmid preparations were unable to activate
the LPS-sensitive CHO/CD14 cells, demonstrating that the plasmids were
not contaminated with LPS.
Transfection and reporter assays
Transient transfections were performed using SuperFect reagent (Qiagen) per the manufacturer’s instructions. Briefly, cells were plated on 6-well dishes 1–2 days before transfection; transfections were performed when cells plated reached 80% confluence. Plasmid DNA was added to 100 µl of Opti-Mem reduced serum media (Life Technologies, Grand Island, NY). All transfections utilized a total of 4 µg of plasmid DNA consisting of 2 µg of reporter plasmid and 1 µg of each expression vector, with the balance made up of the empty vector described above. A total of 10 µl of SuperFect was added to the DNA-medium mixture and incubated for 10 min at ambient temperature. Subsequently, 600 µl of serum-containing medium was added to the reaction mixture and added to the individual wells. Each reaction was prepared individually and each condition was performed in triplicate. Reactions were incubated with the cells for 2–3 h, whereupon the reaction was removed from the cells and fresh media-containing serum was added. On the following day, individual wells were left untreated or were stimulated with either LPS or LAM as indicated in the figures. Cells were then incubated for an additional 5 h before harvesting. Luciferase assays were performed as described below. All transfection experiments were repeated at least three times using different plasmid preparations, and a single representative experiment is shown. Each single experiment represents triplicate independent transfections and data are expressed as average values ± SD.
In transient transfection experiments, immunofluorescence was used to confirm that the transfected cells expressed similar levels of TLR proteins. Briefly, cells were cultured on Costar (Cambridge, MA) chamber slides and transiently transfected with expression plasmids encoding the Flag-TLR fusion protein. Twenty-four hours after transfection, the cells were incubated with a primary anti-FLAG mAb (1 µg/ml; Sigma), and subsequently with a secondary goat anti-mouse Ig antiserum (1:500) conjugated to rhodamine. Transfected cells that expressed the epitope-tagged TLR proteins were counted using a fluorescence microscope. Both CHO cells and RAW264.7 macrophages were found to express similar levels of TLR proteins following transient transfection with the various TLR expression plasmids (data not shown).
Luciferase activity was measured using the Luciferase Assay System
(Promega) according to the manufacturer’s instructions. Briefly, cells
were washed and scraped on ice in cold PBS, pelleted by centrifugation,
and resuspended in 100 µl of reporter lysis buffer. Samples were
freeze-thawed once and centrifuged at 14000 x g for 10
min at 4°C to remove cellular debris. Supernatants were recovered and
assayed for total protein using the Bio-Rad protein assay according to
the manufacturer’s instructions. Fifty micrograms of total protein
from each lysate was assayed for luciferase activity as measured by
light emissions in a scintillation counter. In experiments using 3E10
cells, which contain a stably transfected CD25 reporter gene under the
control of the NF-B-dependent ELAM-1 (E-selectin) promoter, reporter
gene expression was measured by flow cytometry as previously described
(17). Data were collected using CellQuest software (Becton
Dickinson) and were expressed as either mean channel fluorescence
(FL1), or the ratio (fold activation) of the percent of
CD25+ cells in unstimulated and stimulated cell
populations (gated to exclude the lowest 5% of cells based on
mean FL1).
RNA analysis by RT-PCR
Total RNA from 3E10 cells was purified using RNA-STAT (Promega) as recommended by the manufacturer. Reverse transcriptase (RT) reactions to generate cDNA were performed using avian myeloblastosis virus RT (Promega). PCR were performed using Taq polymerase (Promega), 1 µg of cDNA, 0.5 µM of each oligonucleotide primer, 2 mM MgCl2, and 0.2 mM NTPs in a final reaction volume of 50 µl. Thirty amplification cycles were performed (1 min, 94°C denaturation; 1 min, 55°C annealing; 1.5 min, 72°C extension). As a control for contaminating genomic DNA, parallel PCR were performed in which the template nucleic acids were not reverse transcribed. Following amplification, a portion of the PCR were electrophoresed on a 1.2% agarose gel and visualized using ethidium bromide. PCR primers used in this study are as follows: sense strand IL-6 primer, 5'-TTG GGA AAT TTG CCT ACT GAA-3'; anti-sense strand IL-6 primer, 5'-AGG CAT GAC TAT TTT ATC TGG A-3'; sense-strand ß-actin primer, 5'-TCA TGA AGT GTG ACG TTG ACA TCC GT-3'; and antisense strand ß-actin primer, 5'-CCT AGA AGC ATT TGC GGT GCA CGA TG-3'.
Measurement of TNF protein production by stimulated macrophages
Peritoneal exudate macrophages obtained from C3H/FeJ and C3H/HeJ
mice as previously described (18) were stimulated in vitro
with increasing concentrations of LPS or LAM for 6 h. Supernatants
were collected from unstimulated and stimulated cells, centrifuged to
remove cellular debris, and TNF- protein levels were determined
using a specific ELISA (R&D Systems, Minneapolis, MN). Two mice were
used per condition, cells from individual mice were cultured
separately, and ELISA measurements were performed in triplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously proposed that CHO/CD14 cells failed to be activated
by LAM because these cells lacked a CD14-associated signaling protein
that was specifically required to confer LAM responsiveness, but that
was not necessary for LPS responsiveness (16). More
recently, CHO cells were found to not express a functional TLR2 protein
(20). A single nucleotide deletion within the
tlr2 gene of Chinese hamsters, and therefore in CHO cells
derived from these animals, introduced a premature stop codon at aa
520. This mutant gene encodes a putative protein lacking both
transmembrane and intracellular domains of TLR2. This finding supported
the possibility that the missing LAM-responsive signaling protein was
TLR2. To test this hypothesis, a CHO cell line was engineered that was
stably cotransfected with a CD14 expression plasmid and an ELAM-CD25
reporter plasmid (17). A clone of this line (designated
3E10), which constitutively expressed high levels of CD14 on its
surface, was subsequently stably transfected with a Flag-TLR2
expression plasmid. A clone of this line (designated 3E10/TLR2), which
constitutively expressed high levels of Flag on its surface, was
selected for further study. These 3E10/TLR2 cells were stimulated in
vitro for 1 h with LPS, LAM, and IL-1ß in the presence of FBS.
Cell activation was then assessed by measuring the translocation of
NF-B to the nucleus. Following stimulation, the cells were harvested
and nuclear extracts were prepared. EMSA analysis was used to measure
the levels of NF-B in the nuclear extracts. As shown in Fig. 1
, both 3E10 and 3E10/TLR2 cells were
strongly activated by LPS and IL-1ß. In contrast, only the 3E10/TLR2
cells were strongly activated by LAM. These data demonstrate that
expression of functional TLR2 by the 3E10 cells is sufficient to render
these cells responsive to LAM.
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We subsequently sought to determine whether the TLR2-dependent
activation of 3E10 cells by LAM could confer functional responsiveness
on these cells. These cells contain an integrated reporter gene
consisting of the CD25 reporter gene under the NF-B-dependent
regulatory element derived from the ELAM-1 promoter. The capacity of
LAM to activate CD25 expression was assessed by flow cytometry. As
shown in Fig. 2A,
LAM-activated CD25 is expressed in a dose-dependent manner in 3E10/TLR2
cells, whereas 3E10 cells were not activated by LAM to express CD25. In
contrast, both 3E10/TLR2 and 3E10 cells were responsive to LPS, as
judged by CD25 expression (Fig. 2B). Expression of TLR2 only
modestly increased the capacity of these cells to respond to LPS.
Together, these data suggest that TLR2 is an absolute requirement for
LAM responsiveness, whereas TLR2 does not substantially augment LPS
responsiveness in these cells.
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The data presented above demonstrate that expression of functional
TLR2 in the CHO cells can confer LAM responsiveness on these cells, as
judged by the ability to activate NF-B and a NF-B-dependent
reporter gene. We also assessed the capacity of TLR2 to mediate the
activation of an endogenous CHO cell gene in response to LAM treatment.
One of the endogenous CHO genes which can be activated by LPS in
CHO/CD14 cells is IL-6 (21). We used RT-PCR analysis to
determine whether LAM could activate endogenous IL-6 expression in the
3E10/TLR2 cells. As shown in Fig. 3, IL-6
mRNA levels were up-regulated in 3E10 and in 3E10/TLR2 cells stimulated
with either LPS or IL-1ß. In contrast, LAM only up-regulated IL-6
mRNA levels in the 3E10/TLR2 cells. The overexpression of functional
TLR2 in the 3E10/TLR2 did not confer an enhanced ability for LPS to
induce higher levels of IL-6 transcripts compared with levels that were
induced in the 3E10 cells. These findings demonstrate that TLR2 can
specifically mediate the activation of an endogenous gene in CHO cells
by LAM. Also, expression of functional TLR2 does not appear to confer
an enhanced capacity of these cells to respond to LPS.
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A recent study compared the capacities of different TLR proteins
to confer LPS responsiveness on HEK 293 cells. These investigators
reported that TLR2, but not TLR1 or TLR4, could confer LPS
responsiveness on stably transfected HEK 293 cells (9).
This finding contrasted with the conclusion that TLR4 plays the major
role in LPS signaling, based on the demonstration that
LPS-hyporesponsive C3H/HeJ mice possess a point mutation within the
tlr4 gene (11) and that cells from TLR4
knockout mice are also hyporesponsive to LPS (13). To
compare the functional capacities of different TLR proteins, we
utilized a model system in which cells were transiently cotransfected
with expression plasmids encoding the TLR proteins and the ELAM-luc
reporter plasmid. As shown in Fig. 4A, transient cotransfection
of the TLR2 expression plasmid conferred LAM responsiveness on CHO/CD14
cells. In contrast, transiently transfected cells that expressed TLR1
or TLR4 could not be activated by LAM. The responses of these cells to
LPS were qualitatively different from their responses to LAM. In the
CHO/CD14 cells, the TLR4 protein mediated the highest level of LPS
responsiveness, whereas TLR2 could only modestly enhance the
responsiveness of these cells to LPS (Fig. 4B). Expression
of TLR1 did not enhance the responsiveness of these cells to LPS. Thus,
LAM and LPS appear to activate these cells predominantly through
different TLR proteins, specifically TLR2 and TLR4, respectively.
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, C and D, these
cells were capable of activating the ELAM-luc reporter plasmid in
response to LAM and LPS without the overexpression of exogenous TLR
proteins. Furthermore, transient cotransfection of the TLR2 expression
plasmid markedly enhanced the responsiveness of macrophages to LAM. In
contrast, transient transfection with the TLR4 expression plasmid did
not enhance the responsiveness of these cells to LAM. Like CHO/CD14
cells, the responses of macrophages to LPS were qualitatively different
from their responses to LAM. In the RAW264.7 cells, overexpression of
the TLR4 protein substantially enhanced LPS responsiveness, whereas
TLR2 could only modestly enhance the responsiveness of these cells to
LPS relative to control cells. Expression of TLR1 did not enhance the
responsiveness of these cells to LPS or LAM. Thus, like CHO/CD14 cells,
LAM and LPS appear to activate these macrophages predominantly through
TLR2 and TLR4, respectively. Our data provide the direct evidence for
the role of TLR4 in LPS signaling in hamster and murine cells, and are
consistent with the phenotype of the tlr4 mutation in
C3H/HeJ mice. The conserved C-terminal domain of TLR2 is required for cellular activation by LAM
Both Yang et al. (8) and Kirschning et al.
(9) reported that the conserved C-terminal intracellular
domain of TLR2 was required for LPS signaling through TLR2. The
C-terminal 13 amino acids of TLR2 share homology with other members of
the Toll and IL-1 receptor families (6). We used a similar
C-terminal TLR2 mutant to determine whether LAM responsiveness also
required the presence of the C-terminal 13 amino acids. CHO/CD14 cells
were transiently cotransfected with the ELAM-luc reporter plasmid and a
TLR2 expression plasmid, a C-terminal TLR2 mutant expression plasmid
(TLR213), or both expression plasmids. As shown in Fig. 5A, the full-length TLR2 was
capable of conferring LAM responsiveness, whereas the TLR213 mutant
was not. Furthermore, expressing both the full-length and TLR213
mutant proteins in the same cells almost completely blocked the ability
of these cells to be activated by LAM. This finding suggests that the
TLR213 mutant can function as a dominant-negative protein in
LAM signaling and is consistent with an earlier demonstration
that a different C-terminal mutant (TLR2141) can function as a
dominant-negative protein in LPS signaling (8). We
subsequently performed these same experiments using transiently
transfected RAW264.7 macrophages, and similar results were obtained
(Fig. 5B).
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13
mutant proteins in murine RAW264.7 cells did not reduce LAM
responsiveness to levels lower that those of control cells. This
finding differs quantitatively from data obtained using the CHO/CD14
cells, which showed that LAM responsiveness was completely blocked in
cells expressing the TLR213 mutant. These differences can be
explained by the lack of endogenous TLR2 expression in the CHO/CD14
cells. Furthermore, it appears that the TLR213 mutant can
function as a dominant-negative protein in LAM signaling, but only
when blocking signaling through TLR2 from the same species as the
mutant. To test this possibility, we transiently transfected the human
TLR213 mutant into the LPS- and LAM-responsive human THP-1
macrophage-like cell line. As shown in Fig. 5C, we found
that TLR213 suppressed the endogenous capacity of these cells to
respond to LAM, but not to LPS. TLR4-defective C3H/HeJ macrophages respond normally to LAM
Several investigators have demonstrated that C3H/HeJ mice possess
a mutant tlr4 gene in which a single conserved amino acid is
altered (11, 12, 13). These investigators proposed that this
mutation and the mutant protein that is ultimately encoded by this gene
are responsible for the LPS hyporesponsive phenotype of macrophages
obtained from these mice. If this is true, then our data would predict
that the tlr4 mutation in the C3H/HeJ macrophages would have
no effect on the responsiveness of these cells to LAM. Peritoneal
exudate macrophages were obtained from both C3H/HeJ and C3H/FeJ
LPS-responsive mice as described above and then stimulated with LAM or
LPS in vitro. The capacity of the cells to respond to these CD14
ligands was then assessed by measuring TNF- secretion. As shown in
Fig. 6, both LAM- and LPS-induced TNF-
secretion in the C3H/FeJ macrophages, whereas only LAM could induce
substantial TNF- release from the C3H/HeJ macrophages. This finding
agrees with our hypothesis that LAM signaling requires TLR2, but
not TLR4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-dependent promoter and the activation of endogenous
IL-6 gene expression. In contrast to LAM, LPS responsiveness was only
modestly enhanced by overexpression of TLR2 in these cells.
Overexpression of TLR4 did not confer LAM responsiveness on CHO/CD14
cells and did not augment basal LAM responsiveness in RAW264.7
macrophages. Moreover, we provide direct evidence that TLR4 can play a
major role in LPS signaling in both hamster and mouse cells. In
contrast, TLR1 did not appear to mediate cellular activation by either
LPS or LAM. Lastly, we found that macrophages from both LPS-responsive
C3H/FeJ and LPS-unresponsive C3H/HeJ mice were similarly activated by
LAM. Taken together, our data demonstrate that CD14-dependent signaling
induced by LAM and LPS requires distinct TLR proteins. Insights into the mechanism by which TLR proteins generate an intracellular signal in response to these distinct CD14 ligands have come from studying the effects of deletions of the intracellular domain of TLR2 on ligand responsiveness. We found that LAM could not activate CHO/CD14 cells which expressed a mutant TLR2 protein lacking the C-terminal 13 amino acids of the intracellular domain. This domain contains sequences homologous to sequences within the type 1 IL-1 receptor that are required for signal transduction. Furthermore, transient overexpression of this mutant TLR2 protein in either CHO/CD14 cells or the RAW264.7 macrophage cell line did not confer or enhance LAM responsiveness. In the RAW264.7 macrophages, overexpression of the human C-terminal TLR2 mutant did not affect the capacity of endogenous murine TLR2 to mediate LAM responsiveness. In contrast, the C-terminal human TLR2 mutant could function as a dominant-negative mutant when coexpressed with wild-type human TLR2 in both cell lines. Similarly, expression of this human TLR2 mutant in a human monocytic cell line that expresses functional endogenous human TLR2 blocked the activation of these cells by LAM, but not by LPS. This suggests that the ability of TLR2 to form dimers and the ability of mutants to express a dominant-negative phenotype are not shared by TLR proteins from different species. The capacity of C-terminal TLR2 deletion mutants to serve as dominant-negative mutants via their capacity to form complexes with wild-type TLR2 has been recently reported (10), although our findings demonstrate a species-specific feature to this phenotype.
Previous studies have shown that mutation of a single amino acid within
the intracellular domain of TLR4 was sufficient to render macrophages
from C3H/HeJ mice unresponsive to LPS (11, 12, 13). We have
shown in this paper that this mutation did not affect the
responsiveness of these cells to LAM, as judged by the production of
TNF-. This finding is consistent with a previous study by Chatterjee
et al. (22). The LAM responsiveness of CHO/CD14 cells that
overexpress TLR2 was selective, as judged by the inability of LAM
isolated from M. bovis bacillus Calmette-Guérin or
from Mycobacterium tuberculosis to activate the cells
(23). It should be noted that the LAM used in our studies
was purified from a rapidly growing avirulent mycobacterium (AraLAM),
and is chemically distinct from LAM expressed by either M.
bovis bacillus Calmette-Guérin or M. tuberculosis
(ManLAM, reviewed in Ref. 24). Despite this chemical
specificity, several distinct molecules have been reported to activate
cells via TLR2. These include LPS, lipoteichoic acid, and soluble
peptidoglycan (8, 25, 26). Together these data implicate
TLR2 as a pattern recognition receptor for bacterial products.
Several recent publications have demonstrated roles for both TLR2 and TLR4 in LPS signaling. Kirschning et al. (9) reported that overexpression of TLR2, but not TLR4, could confer LPS responsiveness on HEK 293 cells. The same investigators also reported that overexpression of TLR1 did not confer LPS responsiveness on HEK 293 cells (9). A role for TLR2 in LPS responsiveness was also reported by Yang et al. (8) using the same cell line. Both groups of investigators showed that a C-terminal truncation mutant of TLR2 failed to confer LPS responsiveness on the HEK 293 cells. This mutant TLR2 could also function as a dominant-negative mutant in LPS-stimulated human U373 astrocytoma cells (8). In contrast to these studies, a recent paper by Chow et al. (14) reported that TLR4 could also confer LPS responsiveness on HEK 293 cells. These findings are consistent with the capacity of CHO/CD14 cells and of Chinese hamster macrophages that lack functional TLR2 but express functional TLR4 to respond to LPS (20). Moreover, both TLR4-deficient C3H/HeJ mice (11, 12) and TLR4 knockout mice (13) are hyporesponsive to LPS. Thus, while both TLR2 and TLR4 can confer LPS responsiveness when overexpressed in transfected cells, TLR4 appears to be the predominant LPS receptor in vivo. In this paper we have shown that TLR4 is capable of conferring a high level of LPS inducibility on CHO/CD14 cells and that it could further enhance LPS responsiveness in RAW264.7 macrophages. Enhanced LPS responsiveness was not conferred by TLR2. The reasons for these differences are unclear, but might reflect functional or species-specific differences between CHO, RAW264.7, and HEK 293 cells. Furthermore, our functional comparisons of TLR2 and TLR4 were performed in cells that constitutively expressed membrane CD14, whereas the study using HEK 293 cells compared TLR2 and TLR4 function in the absence of membrane CD14 (9). Thus, while both TLR2 and TLR4 may function as LPS receptors, coexpression of membrane CD14 may preferentially enhance the capacity of TLR4 to confer LPS responsiveness. An additional explanation for these differences may also come from the recent finding that an additional protein, MD-2, is required to confer LPS responsiveness on cells that express TLR4 (15). Clonal lines of HEK 293 cells that do not express MD-2 would not be expected to respond to LPS even if these cells overexpress TLR4.
The data presented here demonstrate that the chemically distinct
bacterial glycolipids LAM and LPS can interact with CD14 and activate
cells via different TLR proteins. The dissimilar chemical structures of
LAM and LPS underscore the notion that a variety of bacterial products
can be recognized by CD14, which functions as a pattern recognition
receptor (27) and subsequently activate cells in a
TLR-dependent manner. Our findings suggest a novel receptor-signaling
paradigm in which the binding of distinct ligands (e.g., LAM and LPS)
is mediated by a common receptor chain (CD14) but cellular activation
is initiated via distinct signal-transducing chains that confer ligand
specificity (TLR2 and TLR4). This paradigm contrasts with many cytokine
receptor complexes in which receptor specificity in conferred by a
unique ligand-binding chain, but cellular activation is initiated via
shared signal-transducing chains (e.g., IL-2R and IL-15R). A major
question that remains to be answered is whether different TLR proteins
initiate distinct signaling events or if all TLR proteins utilize a
common signaling pathway. To date, all TLR proteins examined can
activate NF-B and the c-Jun N-terminal kinase/stress-activated
protein kinase pathways via the IRAK and TRAF-6. The recent discovery
of a novel IRAK family member, IRAK-M (28), raises the
possibility that different TLR proteins may preferentially signal
through different IRAK family members.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matthew J. Fenton, Pulmonary Center, R-220, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118-2394. E-mail address:
3 Abbreviations used in this paper: TLR, Toll-like receptor; LAM, lipoarabinomannan; CHO, Chinese hamster ovary; CHO/CD14, CHO cells engineered to express human CD14; FL1, channel fluorescence; RT, reverse transcriptase; LBS, LPS binding protein; ELAM-1, endothelial cell-leukocyte adhesion molecule-1.
Received for publication July 23, 1999. Accepted for publication September 24, 1999.
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M. Muroi, T. Ohnishi, and K.-i. Tanamoto Regions of the Mouse CD14 Molecule Required for Toll-like Receptor 2- and 4-mediated Activation of NF-kappa B J. Biol. Chem., October 25, 2002; 277(44): 42372 - 42379. [Abstract] [Full Text] [PDF]
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T. H. Flo, L. Ryan, E. Latz, O. Takeuchi, B. G. Monks, E. Lien, O. Halaas, S. Akira, G. Skjak-Brak, D. T. Golenbock, et al. Involvement of Toll-like Receptor (TLR) 2 and TLR4 in Cell Activation by Mannuronic Acid Polymers J. Biol. Chem., September 13, 2002; 277(38): 35489 - 35495. [Abstract] [Full Text] [PDF]
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 E. A. Kurt-Jones, L. Mandell, C. Whitney, A. Padgett, K. Gosselin, P. E. Newburger, and R. W. Finberg Role of Toll-like receptor 2 (TLR2) in neutrophil activation: GM-CSF enhances TLR2 expression and TLR2-mediated interleukin 8 responses in neutrophils Blood, August 13, 2002; 100(5): 1860 - 1868. [Abstract] [Full Text] [PDF]
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V. S. Carl, K. Brown-Steinke, M. J. H. Nicklin, and M. F. Smith Jr. Toll-like Receptor 2 and 4 (TLR2 and TLR4) Agonists Differentially Regulate Secretory Interleukin-1 Receptor Antagonist Gene Expression in Macrophages J. Biol. Chem., May 10, 2002; 277(20): 17448 - 17456. [Abstract] [Full Text] [PDF]
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J. Hellman, J. D. Roberts Jr., M. M. Tehan, J. E. Allaire, and H. S. Warren Bacterial Peptidoglycan-associated Lipoprotein Is Released into the Bloodstream in Gram-negative Sepsis and Causes Inflammation and Death in Mice J. Biol. Chem., April 12, 2002; 277(16): 14274 - 14280. [Abstract] [Full Text] [PDF]
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R. van Crevel, T. H. M. Ottenhoff, and J. W. M. van der Meer Innate Immunity to Mycobacterium tuberculosis Clin. Microbiol. Rev., April 1, 2002; 15(2): 294 - 309. [Abstract] [Full Text] [PDF]
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J. J. Watters, J. A. Sommer, Z. A. Pfeiffer, U. Prabhu, A. N. Guerra, and P. J. Bertics A Differential Role for the Mitogen-activated Protein Kinases in Lipopolysaccharide Signaling. THE MEK/ERK PATHWAY IS NOT ESSENTIAL FOR NITRIC OXIDE AND INTERLEUKIN 1beta PRODUCTION J. Biol. Chem., March 8, 2002; 277(11): 9077 - 9087. [Abstract] [Full Text] [PDF]
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T. Vasselon and P. A. Detmers Toll Receptors: a Central Element in Innate Immune Responses Infect. Immun., March 1, 2002; 70(3): 1033 - 1041. [Full Text] [PDF]
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A. Yoshimura, T. Kaneko, Y. Kato, D. T. Golenbock, and Y. Hara Lipopolysaccharides from Periodontopathic Bacteria Porphyromonas gingivalis and Capnocytophaga ochracea Are Antagonists for Human Toll-Like Receptor 4 Infect. Immun., January 1, 2002; 70(1): 218 - 225. [Abstract] [Full Text] [PDF]
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P. Henneke, O. Takeuchi, J. A. van Strijp, H.-K. Guttormsen, J. A. Smith, A. B. Schromm, T. A. Espevik, S. Akira, V. Nizet, D. L. Kasper, et al. Novel Engagement of CD14 and Multiple Toll-Like Receptors by Group B Streptococci J. Immunol., December 15, 2001; 167(12): 7069 - 7076. [Abstract] [Full Text] [PDF]
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P. I. Song, T. A. Abraham, Y. Park, A. S. Zivony, B. Harten, H. F. Edelhauser, S. L. Ward, C. A. Armstrong, and J. C. Ansel The Expression of Functional LPS Receptor Proteins CD14 And Toll-Like Receptor 4 in Human Corneal Cells Invest. Ophthalmol. Vis. Sci., November 1, 2001; 42(12): 2867 - 2877. [Abstract] [Full Text] [PDF]
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B. Pulendran, P. Kumar, C. W. Cutler, M. Mohamadzadeh, T. Van Dyke, and J. Banchereau Lipopolysaccharides from Distinct Pathogens Induce Different Classes of Immune Responses In Vivo J. Immunol., November 1, 2001; 167(9): 5067 - 5076. [Abstract] [Full Text] [PDF]
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 B W Jones, K A Heldwein, T K Means, J J Saukkonen, and M J Fenton Differential roles of Toll-like receptors in the elicitation of proinflammatory responses by macrophages Ann Rheum Dis, November 1, 2001; 60(90003): iii6 - 12. [Abstract] [Full Text] [PDF]
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I. C. Almeida and R. T. Gazzinelli Proinflammatory activity of glycosylphosphatidylinositol anchors derived from Trypanosoma cruzi: structural and functional analyses J. Leukoc. Biol., October 1, 2001; 70(4): 467 - 477. [Abstract] [Full Text] [PDF]
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A. Schonemeyer, R. Lucius, B. Sonnenburg, N. Brattig, R. Sabat, K. Schilling, J. Bradley, and S. Hartmann Modulation of Human T Cell Responses and Macrophage Functions by Onchocystatin, a Secreted Protein of the Filarial Nematode Onchocerca volvulus J. Immunol., September 15, 2001; 167(6): 3207 - 3215. [Abstract] [Full Text] [PDF]
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A. E. Medvedev, P. Henneke, A. Schromm, E. Lien, R. Ingalls, M. J. Fenton, D. T. Golenbock, and S. N. Vogel Induction of Tolerance to Lipopolysaccharide and Mycobacterial Components in Chinese Hamster Ovary/CD14 Cells Is Not Affected by Overexpression of Toll-Like Receptors 2 or 4 J. Immunol., August 15, 2001; 167(4): 2257 - 2267. [Abstract] [Full Text] [PDF]
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S. Sugawara, S. Yang, K. Iki, J. Hatakeyama, R. Tamai, O. Takeuchi, S. Akashi, T. Espevik, S. Akira, and H. Takada Monocytic Cell Activation by Nonendotoxic Glycoprotein from Prevotella intermedia ATCC 25611 Is Mediated by Toll-Like Receptor 2 Infect. Immun., August 1, 2001; 69(8): 4951 - 4957. [Abstract] [Full Text] [PDF]
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C. Neufert, R. K. Pai, E. H. Noss, M. Berger, W. H. Boom, and C. V. Harding Mycobacterium tuberculosis 19-kDa Lipoprotein Promotes Neutrophil Activation J. Immunol., August 1, 2001; 167(3): 1542 - 1549. [Abstract] [Full Text] [PDF]
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M. T. Abreu, P. Vora, E. Faure, L. S. Thomas, E. T. Arnold, and M. Arditi Decreased Expression of Toll-Like Receptor-4 and MD-2 Correlates with Intestinal Epithelial Cell Protection Against Dysregulated Proinflammatory Gene Expression in Response to Bacterial Lipopolysaccharide J. Immunol., August 1, 2001; 167(3): 1609 - 1616. [Abstract] [Full Text] [PDF]
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O. Takeuchi, T. Kawai, P. F. Muhlradt, M. Morr, J. D. Radolf, A. Zychlinsky, K. Takeda, and S. Akira Discrimination of bacterial lipoproteins by Toll-like receptor 6 Int. Immunol., July 1, 2001; 13(7): 933 - 940. [Abstract] [Full Text] [PDF]
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J. Nigou, C. Zelle-Rieser, M. Gilleron, M. Thurnher, and G. Puzo Mannosylated Lipoarabinomannans Inhibit IL-12 Production by Human Dendritic Cells: Evidence for a Negative Signal Delivered Through the Mannose Receptor J. Immunol., June 15, 2001; 166(12): 7477 - 7485. [Abstract] [Full Text] [PDF]
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B. W. Jones, T. K. Means, K. A. Heldwein, M. A. Keen, P. J. Hill, J. T. Belisle, and M. J. Fenton Different Toll-like receptor agonists induce distinct macrophage responses J. Leukoc. Biol., June 1, 2001; 69(6): 1036 - 1044. [Abstract] [Full Text] [PDF]
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A. A. Chackerian, T. V. Perera, and S. M. Behar Gamma Interferon-Producing CD4+ T Lymphocytes in the Lung Correlate with Resistance to Infection with Mycobacterium tuberculosis Infect. Immun., April 1, 2001; 69(4): 2666 - 2674. [Abstract] [Full Text] [PDF]
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S. Shoham, C. Huang, J.-M. Chen, D. T. Golenbock, and S. M. Levitz Toll-Like Receptor 4 Mediates Intracellular Signaling Without TNF-{{alpha}} Release in Response to Cryptococcus neoformans Polysaccharide Capsule J. Immunol., April 1, 2001; 166(7): 4620 - 4626. [Abstract] [Full Text] [PDF]
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M. M. Monick, A. B. Carter, P. K. Robeff, D. M. Flaherty, M. W. Peterson, and G. W. Hunninghake Lipopolysaccharide Activates Akt in Human Alveolar Macrophages Resulting in Nuclear Accumulation and Transcriptional Activity of {{beta}}-Catenin J. Immunol., April 1, 2001; 166(7): 4713 - 4720. [Abstract] [Full Text] [PDF]
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T. K. Means, B. W. Jones, A. B. Schromm, B. A. Shurtleff, J. A. Smith, J. Keane, D. T. Golenbock, S. N. Vogel, and M. J. Fenton Differential Effects of a Toll-Like Receptor Antagonist on Mycobacterium tuberculosis-Induced Macrophage Responses J. Immunol., March 15, 2001; 166(6): 4074 - 4082. [Abstract] [Full Text] [PDF]
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 G.A.W. Rook, G. Seah, and A. Ustianowski M. tuberculosis: immunology and vaccination Eur. Respir. J., March 1, 2001; 17(3): 537 - 557. [Abstract] [Full Text] [PDF]
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T. H. Flo, O. Halaas, S. Torp, L. Ryan, E. Lien, B. Dybdahl, A. Sundan, and T. Espevik Differential expression of Toll-like receptor 2 in human cells J. Leukoc. Biol., March 1, 2001; 69(3): 474 - 481. [Abstract] [Full Text]
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T. Kikuchi, T. Matsuguchi, N. Tsuboi, A. Mitani, S. Tanaka, M. Matsuoka, G. Yamamoto, T. Hishikawa, T. Noguchi, and Y. Yoshikai Gene Expression of Osteoclast Differentiation Factor Is Induced by Lipopolysaccharide in Mouse Osteoblasts Via Toll-Like Receptors J. Immunol., March 1, 2001; 166(5): 3574 - 3579. [Abstract] [Full Text] [PDF]
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D. Chakravortty, Y. Kato, T. Sugiyama, N. Koide, M. M. Mu, T. Yoshida, and T. Yokochi Inhibition of p38 Mitogen-Activated Protein Kinase Augments Lipopolysaccharide-Induced Cell Proliferation in CD14-Expressing Chinese Hamster Ovary Cells Infect. Immun., February 1, 2001; 69(2): 931 - 936. [Abstract] [Full Text] [PDF]
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G. R. Strohmeier, J. H. Walsh, E. S. Klings, H. W. Farber, W. W. Cruikshank, D. M. Center, and M. J. Fenton Lipopolysaccharide Binding Protein Potentiates Airway Reactivity in a Murine Model of Allergic Asthma J. Immunol., February 1, 2001; 166(3): 2063 - 2070. [Abstract] [Full Text] [PDF]
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A. M. Hajjar, D. S. O'Mahony, A. Ozinsky, D. M. Underhill, A. Aderem, S. J. Klebanoff, and C. B. Wilson Cutting Edge: Functional Interactions Between Toll-Like Receptor (TLR) 2 and TLR1 or TLR6 in Response to Phenol-Soluble Modulin J. Immunol., January 1, 2001; 166(1): 15 - 19. [Abstract] [Full Text] [PDF]
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S. Tsuji, M. Matsumoto, O. Takeuchi, S. Akira, I. Azuma, A. Hayashi, K. Toyoshima, and T. Seya Maturation of Human Dendritic Cells by Cell Wall Skeleton of Mycobacterium bovis Bacillus Calmette-Guerin: Involvement of Toll-Like Receptors Infect. Immun., December 1, 2000; 68(12): 6883 - 6890. [Abstract] [Full Text] [PDF]
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E. Cario and D. K. Podolsky Differential Alteration in Intestinal Epithelial Cell Expression of Toll-Like Receptor 3 (TLR3) and TLR4 in Inflammatory Bowel Disease Infect. Immun., December 1, 2000; 68(12): 7010 - 7017. [Abstract] [Full Text] [PDF]
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T. Matsuguchi, T. Musikacharoen, T. Ogawa, and Y. Yoshikai Gene Expressions of Toll-Like Receptor 2, But Not Toll-Like Receptor 4, Is Induced by LPS and Inflammatory Cytokines in Mouse Macrophages J. Immunol., November 15, 2000; 165(10): 5767 - 5772. [Abstract] [Full Text] [PDF]
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R. I. Tapping, S. Akashi, K. Miyake, P. J. Godowski, and P. S. Tobias Toll-Like Receptor 4, But Not Toll-Like Receptor 2, Is a Signaling Receptor for Escherichia and Salmonella Lipopolysaccharides J. Immunol., November 15, 2000; 165(10): 5780 - 5787. [Abstract] [Full Text] [PDF]
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M. Fujihara, S. Wakamoto, T. Ito, M. Muroi, T. Suzuki, H. Ikeda, and K. Ikebuchi Lipopolysaccharide-triggered desensitization of TNF-{alpha} mRNA expression involves lack of phosphorylation of I{kappa}B{alpha} in a murine macrophage-like cell line, P388D1 J. Leukoc. Biol., August 1, 2000; 68(2): 267 - 276. [Abstract] [Full Text]
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