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Functional Genomics Group,
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Division of Molecular and Genetic Medicine,
Sheffield Hybridomas, University of Sheffield, United Kingdom; and
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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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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At least six mammalian TLRs have been characterized (4, 5), and it seems likely that others remain to be identified. It is clear from genetic, biochemical, and Ab blockade experiments that the physiological roles of the two best-characterized TLRs, TLR2 and TLR4, differ: TLR4 is required for in vivo LPS responsiveness, whereas TLR2 is required for responsiveness to microbial products other than LPS. TLR2 agonists include lipoproteins (6, 7, 8, 9). The current data are compatible with a model in which TLRs are receptors for conserved microbial determinants, while other pattern recognition receptors, such as CD14, lack signaling domains but present microbial determinants to TLRs (10, 11, 12).
The functional form of transmembrane receptors commonly comprises several different proteins, which form heteromeric complexes. Individual components may express poorly on their own (13, 14). Binding and signaling functions may be subserved by different proteins of the complex. An example of this is the mammalian T and B cell Ag receptors, in which covalently linked binding chains engage ligand (15), while other receptor chains are required for signaling, but not for ligand binding (16). In other cases, although binding and signaling domains may exist on the same protein, ligand specificity is determined by combinations of multiple receptor chains, as seen in the cytokine (17, 18) and TGF-ß receptor families (19).
Heterodimerization is important in the IL-1R family, which, like the TLR family, contains receptors with TIR signaling domains. IL-1 and IL-18 signaling is mediated not by a single IL-1R family member, but by a heterodimer consisting of two family members. Only one chain is a high affinity cytokine binding protein (20, 21), although the accessory member of the heterodimer is essential for cytokine signaling (22, 23). Accessory proteins are structurally very similar to other family members, and their accessory role cannot be distinguished from that of other IL-1R family members by primary sequence analysis.
In this study we observed that responsiveness to some products of Gram-negative bacteria by transfected HeLa cells is influenced by coexpression of both TLR2 and TLR1, a TLR of previously unknown function. Using an mAb generated against TLR1, we showed that TLR1, like TLR2 (10), is expressed in human mononuclear cells and propose a model involving functional heterodimerization between TLRs to explain the cooperative effect observed between TLR1 and TLR2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA was extracted from HeLa cells and from Ficoll-Hypaque (Lymphoprep, Nycomed)-purified human PBMC using RNAzol B (Biogenesis, Bournemouth, U.K.). cDNA was made using AMV retrotranscriptase (Promega, Madison, WI) with oligo(dT) priming, according to the manufacturer’s recommendations. Controls without retrotranscription were also generated.
Vectors
pCMV-TLR1.
A clone of the human TLR1 open reading frame was obtained from human
PBMC cDNA by PCR using Pfu polymerase (Stratagene, La Jolla, CA) and a
TA cloning kit (TA-TOPO, Invitrogen, San Diego, CA). Primers are shown
in Table I
. Three independent TLR1
clones, obtained by PCR from PBMC cDNA of a single individual, were
sequenced. The sequence found differs from that published (GenBank
D13637), having a G at position 302. This codon (AGA) specifies R (not
T). HinfI restriction of TLR1 fragments amplified from PBMC
cDNA from healthy volunteers confirmed that the sequence at position
302 of our clone was also present in all six individuals examined (data
not shown). pCMV-TLR1 was generated by inserting the cloned full-length
TLR1 open reading frame into pCDNA3 (Invitrogen).
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cyt.
pCMV-TLR1cyt encoding only the extracellular and transmembrane
portions of TLR1 was generated by joining the
Asp718I-HindIII fragment of pCMV-TLR1
with a HindIII-XbaI-digested mutant TLR1
fragment. PCR mutagenesis with primers
5'-TCAGATGTTTACCTCCCAGGATCAAG and the reverse mutagenic primer
5'-TCTAGATTATTACCTGCGCCGGGTCTGGGTCCACTG-3' was used to insert stop
codons after the stop transfer signal RRRAR.
pFLAG-TLR2.
The TLR2 open reading frame was amplified as described for TLR1.
Primers used are shown in Table I
. The product has the sequence
previously described (24). pFLAG-TLR2 is the amplified
TLR2 NotI-XbaI fragment subcloned into pFLAG-CMV1
(Sigma, St. Louis, MO), which replaces TLR2 aa 1–17 (24)
with a preprotrypsin leader and FLAG epitope.
Other vectors.
pFLAG-TLR4 vector (pFLAG-hToll) (25) was a gift from Dr.
M. Muzio (Instituto Medic Negri, Milan, Italy). pFLAG-MD2 was
constructed as described for TLR2, using primers shown in shown in
Table I. The amplified sequence found was identical with that described
previously (26). pFLAG-IL-1RAcP, which produces
flag-tagged IL-1R accessory protein (27), was a gift from
Dr. F. Volpe, Glaxo-Wellcome (Stevenage, U.K.). pCDM8-CD14
(28) was a gift from Prof. B. Seed (Massachusetts General
Hospital, Boston, MA). pPL-pLUC consists of the -174 to +45 region of
the human IL-8 gene (GenBank M28130, bases 1310–1645) subcloned into
the firefly luciferase vector pGL3-Basic (Promega), creating
pIL-8-pLuc. pTK-rLUC was obtained from Promega. Plasmid identities were
confirmed by restriction mapping; the sequences of all constructs and
mutants constructed here were confirmed by sequencing. All plasmids
were prepared using Qiagen endotoxin-free Maxiprep kits (Valencia,
CA).
TLR1Fc production
An XhoI site was introduced by PCR into the TLR1
sequence at the predicted extracellular-transmembrane junction. The
TLR1Fc expression vector pCMV-TLR1Fc was constructed by ligation of the
TLR1 extracellular domain into a pCDNA3 vector containing an engineered
XhoI site 5' to the hinge of the human IgG1 heavy chain. The
predicted amino acid sequence of the junction is SELSCNSSGDKKV. A
TLR1Fc-producing NSO/1 murine myeloma line was produced as previously
described (29). Briefly, cells were electroporated with
pCMV-TLR1Fc, selected with 1 mg/ml G418 (Life Technologies,
Gaithersburg, MD), and screened for Fc production. A secreting clone
was established, subcloned twice, and grown in Spinner cultures in RPMI
plus 1% low IgG FBS (both from Life Technologies). TLR1Fc was purified
from batches of 20–30 liters of conditioned medium by protein A
affinity chromatography. Elution with 25 mM MOPS and 4 M
MgCl2 (pH 6.0) was followed by gel filtration
into PBS (pH 7.2) and freezing at -80°C. Purity, as assessed by
SDS-PAGE, was >95%. Concentrations of TLR1Fc were determined with a
human Fc
-specific sandwich ELISA. This used specific polyclonal goat
anti-human Fc antisera (Jackson ImmunoResearch Laboratories,
West Grove, PA) and was standardized with human IgG1 myeloma protein
(Calbiochem, Torrance, CA).
mAb production
Using standard techniques, mice were immunized repeatedly with TLR1Fc. Following splenocyte fusion with the partner NSO/1, hybridoma supernatants were screened for anti-TLR1Fc reactivity by ELISA and for reactivity with TLR1-transfected HeLa cells by flow cytometry as described below. Here we describe one reactive clone, GD2.F4, of isotype IgG1, as determined by an isotyping kit (Serotec, Oxford, U.K.).
HeLa cell culture
HeLa cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM (Life Technologies) plus 10% FBS. All FBS used was heat treated for 30 min at 56°C. The medium and serum were endotoxin free, as tested by the manufacturer. All cell lines used tested negative for mycoplasma.
Analysis of TLR mRNA expression in HeLa cells and PBMC
Expression of TLR1, TLR2, TLR4, MD-2, and the housekeeping gene
encoding the ribosomal L41- homologue protein (GenBank Z12962) was
analyzed in PBL and HeLa cells. The retrotranscript of 40 ng of total
RNA was used as template for PCR with Taq polymerase (Promega).
Conditions and primers used are shown in shown in Table I. The
TLR4 primers span the apparent unexcised intron in GenBank sequence
U88880, but not U93091.
Flow cytometry of HeLa cells
HeLa cells were grown in 100-mm dishes to 50% confluence and transfected with pCMV-TLR1 (10 µg) using the calcium phosphate method (30). For comparison of expression levels on cotransfection of different TLRs, 5 µg each of TLR expression vector or pCDNA3 were used. Control transfections with a pCMV-enhanced green fluorescent protein vector showed >70% transfection efficiency under these conditions. Thirty-six hours after transfection cells were washed off the plate using PBS and 5 mM EDTA (pH 7.2) and resuspended in staining buffer (PBS, 5% goat serum, and 0.1% sodium azide) on ice. mAb GD2.F4 or IgG1 anti-keyhole limpet hemocyanin isotype control (PharMingen, San Diego, CA) was added to 5 µg/ml. A second layer consisted of staining buffer containing 3 µg/ml biotinylated goat anti-mouse IgG (Jackson ImmunoResearch). The third layer was staining buffer containing streptavidin-PE (Sigma; 10%, v/v). Cells were washed twice between each step. Fluorescence was measured with a FACSCalibur cytometer (Becton Dickinson, Mountain View, CA) with logarithmic FL2 amplifier.
Flow cytometry of mononuclear cells
Mononuclear cells from venous blood of healthy donors were purified by centrifugal elutriation. The monocyte population was identified by size gating, and purity was confirmed by anti-HLA-DR and anti-CD14 immunostaining. One hundred percent of the monocytes expressed these markers (data not shown). Cells (5 x 105) were suspended in staining buffer (PBS, 0.1% BSA, and 0.02% sodium azide) with 100 µg/ml human IgG and 15 µg/ml GD2.F4 or isotype control. Secondary Ab was a one-eighth dilution of goat anti mouse IgG F(ab')2-FITC conjugate (Roche, Indianapolis, IN). Cells were washed twice with staining buffer between each step and were analyzed by flow cytometry.
Bacterial growth and LPS preparations
Meningitidis strain H44/76 (pLAK33) was derived from wild-type
clinical isolate H44/76 by targeted disruption of the lpxA
gene as previously described (31). N.
meningitidis H44/76 LPS, isolated by the hot phenol extraction
method (32), was a gift from Dr. P. van der Ley (National
Institute of Public Health and Environment, Bilthoven, The
Netherlands). Highly purified Escherichia coli K235 LPS,
prepared using a modified extraction procedure (33), was a
gift of Dr. S. Vogel (Uniformed Services University of the Health
Sciences, Bethesda, MD). Other LPS preparations used were purified by
phenol/water extraction and purchased from Sigma (Gillingham, Dorset,
U.K.). Stock solutions were stored dissolved in PBS at 5 mg/ml. Soluble
factors released by live N. meningitidis were collected as
follows. Soluble factors released by N. meningitidis were
collected by growing H44/76 N. meningitidis in
Mueller-Hinton broth (Oxoid; Basingstoke, Kent, U.K.) to mid-log phase,
pelleting, washing twice in DMEM, and resuspending at 
2 x
108 CFU/ml in a 5-ml sterile endotoxin-free
Sterilin (Stone, Staffs, U.K.) vessel. After incubation at 200 rpm at
37°C in 5% CO2 for 1 h, conditioned
culture medium was filtered using Acrodisc 0.2-µm pore size filters
(Gelman, Oberlin, OH).
Transfection and reporter assays in HeLa cells
HeLa cells (ECACC, 85060701; 1.5 x 104/well) were seeded into 96-well tissue culture plates 24 h before transfection. Transfections were performed using SuperFect (Qiagen) according to the manufacturer’s advice; each well received 25 ng of pCDM8-CD14, 400 ng of pPL-IL-8, 100 ng of pTK-rLuc for normalization of transfection efficiency, and 50 ng of the TLR-expressing vector under test. Sufficient pcDNA3 (Invitrogen; empty vector) was added to keep the total DNA dose constant. After transfection, cells were washed, and 100 µl of fresh medium was added. Triplicate wells were transfected for each treatment. Twenty-four hours later, agonists were added. These were prepared and added as 10x stock solutions in PBS. Thirty-six hours following transfection reporter levels were measured using the dual luciferase system (Promega) as recommended by the manufacturer. Normalized IL-8 promoter activity is the ratio of firefly to Renilla luciferase activity.
IL-8 ELISA
HeLa cells were transfected as described above, except that 500 ng of pCDNA3 was used in place of the reporter vectors, and 200 µl of medium was added following transfection. Twenty-four hours later 100 µl was aspirated, and agonists were added as 20x solutions in 5 µl of PBS. Twelve hours subsequently the media were aspirated. After removal of any cellular material by centrifugation, media were stored at -20°C until assayed. IL-8 immunoreactivity was determined using specific polyclonal sheep anti-IL-8 antisera (generated and donated by Dr. S. Poole, National Institute for Biological Standards and Control, Potter’s Bar, U.K.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To define which subpopulations in human peripheral blood
leukocytes express TLR1, monoclonal antibodies were generated against
recombinant soluble TLR1 extracellular domain (TLR1-Fc). TLR1Fc was
produced in a murine myeloma line as a fusion to the human IgG1 Fc
region. The protein migrates as two major bands on nonreducing SDS-PAGE
gels (Fig. 1A) and one smaller
band on reducing gels (Fig. 1B). This may be due to
different patterns of disulfide bonding. Sequencing of the band
migrating faster on nonreducing gels showed the amino terminus of the
recombinant protein to be SEFLVDRSKN. This corresponds to aa 25 of the
predicted propeptide and is three amino acids from the predicted
signalase cleavage site (SigCleave, Genetics Computer Group, Madison,
WI). TLR1Fc was used as an immunogen to generate mAb against TLR1; Ab
GD2.F4 does not recognize human IgG1, but recognizes both TLR1Fc forms
(data not shown). It specifically affinity purifies both forms of
TLR1Fc from TLR1Fc containing tissue culture medium (data not shown).
It recognizes HeLa cells transfected with TLR1 expression vectors (Fig. 2A), but does not bind to mock
transfected HeLa, a cell line that lacks TLR1 mRNA (see below). Higher
surface GD2 epitope expression is seen in cells transfected with the
deletion construct pCMV-TLR1cyt than with the full-length construct
pCMV-TLR1 (Fig. 2A). No binding of isotype control Ab to
transfected HeLa cells was observed (data not shown). The specificity
of GD2.F4 was tested by staining cells transfected with other TLRs. It
does not recognize TLR2, TLR4, or TLR4 and MD2 (Fig. 2B)
(Table II).
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shows that all cells in a monocyte
population, purified from the blood of healthy volunteers by
elutriation, express TLR1. A more detailed analysis will be reported
elsewhere (A. Visintin et al., manuscript in preparation).
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The effect of TLR1 on responses to bacteria was investigated. The
cell line HeLa was used, because this cell line does not express
detectable TLR1 or TLR2 messages (Fig. 4). Although TLR4 message is detectable,
message for MD-2, a protein required for TLR4 function
(26), is not (Fig. 4). We used luciferase controlled by an
IL-8 promoter fragment as a reporter. This reporter responds to LPS in
RAW264.7 cells (data not shown). The cell culture medium used included
10% FBS, a source of LPS-binding protein (34). We
consistently observed, as previously reported (11, 35),
that cells transfected with TLR1 alone did not respond to E.
coli or N. meningitidis bacteria (data not shown).
However, Fig. 5A shows that in
cells transfected with TLR1 and TLR2, induction of the IL-8 promoter
was elicited by soluble factors released by a growing wild-type H44/76
organism. Efficient induction depended on transfection of both TLR1 and
TLR2 (Fig. 5B).
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Cells transfected with TLR2 have been reported to respond to LPS
preparations (11, 24). LPS is released by growing N.
meningitidis (36); thus, the structure recognized by
TLR2 and TLR1 in the Neisseria supernatants might be LPS.
Therefore, the stimulatory effect of soluble factors released from
mid-log phase wild-type N. meningitidis strain H44/76 and
from its LPS-free isogenic mutant pLAK33 (31), prepared as
described in Materials and Methods, were
compared. An LPS preparation, prepared by phenol-water extraction from
strain H44/76, was also studied. Fig. 5B shows that
induction of the IL8 gene depended on transfection of both TLR1 and
TLR2 and on the presence of N. meningitidis LPS.
Compatible with results from observations with other LPS preparations
used in this study (below), none of the Neisseria
preparations tested caused IL-8 promoter induction in cells transfected
with TLR1 alone (data not shown).
TLR1/2 agonist activity resides in only some LPS preparations
The influence of TLR1/2 cotransfection on responses of HeLa cells
to other preparations of LPS was investigated, because copurification
of other bacterial products with LPS can contribute significantly to
bioactivity of LPS preparations (37). N.
meningitidis contains LPS of the rough type (38);
signaling to another rough LPS preparation, from S.
minnesota Re595, was also dependent on both TLR1 and TLR2. The
increases in IL-8 promoter activity induced by S. minnesota
Re595 LPS preparation in four independent experiments are summarized in
Table III; a similar pattern of responses
was observed when IL-8 production was measured directly by ELISA (Table III), confirming that the reporter and the genomic IL-8 gene behave
similarly. By contrast, the responses to two smooth LPS preparations
were not affected by TLR1 (Fig. 6).
Likewise, responses to a highly repurified E. coli K235 LPS
preparation (a gift from Dr. S. Vogel) did not depend on TLR1. Note
that weak stimulation via TLR2 was observed at high doses in this
system (Fig. 7). By contrast, when HeLa
cells were transfected with an expression vector producing MD-2, a
component of the TLR4 receptor complex that is not expressed in HeLa
cells (Fig. 4), the repurified LPS produced a strong response (Fig. 7).
The effect of TLR1 cotransfection was not due to alteration in cell
surface expression of TLR2 (Table II), nor was it bypassed by
increasing the TLR2 plasmid dose (data not shown). The TLR1/2
cooperative effect did not depend on whether the Re595 stock solution
was dissolved in PBS or in PBS/0.2% triethylamine as previously
described (39) (data not shown).
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To investigate the mechanism of the observed TLR1-TLR2
cooperation, TLR2 was cotransfected with vectors directing expression
of TLR1, TLR1cyt, or the accessory chain of the IL-1R (IL-1RAcP)
(27), which contains a TIR signaling domain related to
those of TLRs (3). We reasoned that if the increase in
signaling was a nonspecific effect due to increased expression of TIR
domains, then IL-1RAcP might substitute for TLR1. Flow cytometry showed
IL-1RAcP to be expressed at the cell surface in HeLa (data not shown).
Despite cell surface expression of both TLR1cyt (Fig. 2) and
IL-1RAcP (data not shown), neither could substitute for full-length
TLR1 in the cooperativity effect (Fig. 8). Thus, the intracellular domain of
TLR1 is required for cooperativity with TLR2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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66% complete), suggesting that there may be 25–30
TLRs in the complete genome. Even if combinatorial function is limited
to dimerization, this would generate 500–1,000 different receptors if
any pair is functional. Further, similar database searching shows that
there are least six MDs (MD-1, MD-2, and four others) in the human
genome. If, as current data suggest, these are sensitizers/modifiers
for TLR function, then a further diversification into the 1–10,000
range is potentially possible. This report leaves several issues unresolved. The first is whether TLR2 is the physiological partner of TLR1. However, Muzio and colleagues have recently shown that TLR1 mRNA, or a closely related sequence, is expressed on all leukocyte subsets analyzed, as judged by Northern blotting (43). We demonstrate TLR1 monocyte surface expression in this work; TLR2 expression by monocytes has previously been reported (10). Therefore, TLR1 and TLR2 are coexpressed in normal human monocytes. However, we cannot exclude the possibility that, as occurs with components of several multichain cytokine receptor families (18, 44), TLR1 interacts functionally with other molecules as well. Study of the phenotype of mice with targeted deletions of TLR1 will help resolve this issue.
Abundant data are now available to show that in vivo responses to LPS depend on TLR4, not TLR2. This has been shown by genetic means (7, 45), by Ab blockade of TLR4 (9), and by demonstration that species-specific variation in LPS structures recognition can be reproduced by transfection of cell lines with TLR4 clones from that species (12). By contrast, LPS responses of TLR2 knockout animals appear normal (6). Rather, TLR2 responds to synthetic lipoproteins in a stereospecific fashion (46), and lipoprotein effects on cell lines can be blocked by mAbs to TLR2 (47). TLR2 responses to several other bacterial preparations of less defined composition have also been described (35, 48, 49, 50). Therefore, a second unresolved issue concerns the physiological nature of the TLR1/2 agonist. Although we show that such an entity exists in some LPS preparations and is secreted by N. meningitidis in an LpxA-dependent fashion, the precise nature of the agonist is not known. Previous work has shown that LPS preparations from rough Salmonella mutants include proteins, and perhaps other cell wall constituents, that remain tightly associated with LPS during conventional phenol extraction (37). These factors act via TLR4-independent receptor systems (33) and contribute to the bioactivity of the crude LPS preparation, acting synergistically with the LPS, which is absolutely dependent on TLR4 for its effect
The presence of the TLR1/2 agonist in only some LPS preparations
suggests that it is unlikely to be LPS per se, a conclusion supported
by the weak activity of a repurified LPS preparation in the
TLR2-transfected or TLR1- and TLR2-transfected cells and compatible
with the demonstration of normal LPS responsiveness in TLR2 knockout
animals (6, 51). Genetic analysis of genes required for
secretion of TLR1/2 agonist by N. meningitidis showed
requirement for LpxA, a gene that encodes part of the LPS
biosynthesis pathway of Gram-negative organisms (52). This
suggests that the TLR1/2 agonist is metabolically related to LPS or
requires LPS for efficient expression or secretion, as would be the
case if the TLR1/2 agonist were a lipoprotein attached to LPS in a
complex. There is evidence that such complexes exist and that LPS and
the other associated component(s) act synergistically to activate
macrophages (33, 37). Our data show that HeLa cells
express TLR4, but not MD-2, and therefore do not respond to LPS alone
(Figs. 4 and 7), but become highly LPS responsive when transfected with
MD2 and CD14, but not with CD14 alone (Fig. 7). Therefore, it is
possible that when HeLa cells are transfected with TLR1 and TLR2
(+CD14), either TLR1/TLR2 form a receptor that responds to such a
complex agonist or, alternatively, TLR1/TLR2 mediate responses to a
specific agonist and a separate LPS signal (subthreshold by itself,
without MD-2) is mediated by TLR4, both being required for detectable
effects in our assays. Detailed biochemical studies and a greater
understanding of the extent of functional cooperation between different
TLR family members (42) will be required to resolve these
issues. Finally, given the findings alluded to earlier that up to 30
TLRs and six MDs may be encoded in the human genome, some caution must
be exercised in interpreting the data on TLR function published to
date, because any cell line or population may express multiple TLRs and
MDs not yet accounted for that contribute to the observed pattern of
responses. Further, interpretation of studies on knockout and mutant
mice become more complex if functional responses to pathogens and their
products are mediated by oligomeric structures containing two or more
TLRs and/or MDs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Prof. Steven Dower, Functional Genomics Group, Division of Molecular and Genetic Medicine, University of Sheffield, Royal Hallamshire Hospital, M Floor, Sheffield, U.K. S10 2JF.
3 Abbreviations used in this paper: TLR, Toll-like receptor; TIR, Toll and IL-1 receptor related.
Received for publication January 5, 2000. Accepted for publication September 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and JNK/SAPK activation upstream of tumor necrosis factor receptor-associated factor 6 (TRAF6). J. Exp. Med. 187:2097.-chain cytoplasmic domains is required for signalling. Nature 369:330.[Medline]
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 B. Schmeck, S. Huber, K. Moog, J. Zahlten, A. C. Hocke, B. Opitz, S. Hammerschmidt, T. J. Mitchell, M. Kracht, S. Rosseau, et al. Pneumococci induced TLR- and Rac1-dependent NF-{kappa}B-recruitment to the IL-8 promoter in lung epithelial cells Am J Physiol Lung Cell Mol Physiol, April 1, 2006; 290(4): L730 - L737. [Abstract] [Full Text] [PDF]
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J.-S. Chang, J. F. Huggett, K. Dheda, L. U. Kim, A. Zumla, and G. A. W. Rook Myobacterium tuberculosis Induces Selective Up-Regulation of TLRs in the Mononuclear Leukocytes of Patients with Active Pulmonary Tuberculosis. J. Immunol., March 1, 2006; 176(5): 3010 - 3018. [Abstract] [Full Text] [PDF]
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P. Massari, A. Visintin, J. Gunawardana, K. A. Halmen, C. A. King, D. T. Golenbock, and L. M. Wetzler Meningococcal Porin PorB Binds to TLR2 and Requires TLR1 for Signaling J. Immunol., February 15, 2006; 176(4): 2373 - 2380. [Abstract] [Full Text] [PDF]
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 C. M. O'Connell, I. A. Ionova, A. J. Quayle, A. Visintin, and R. R. Ingalls Localization of TLR2 and MyD88 to Chlamydia trachomatis Inclusions: EVIDENCE FOR SIGNALING BY INTRACELLULAR TLR2 DURING INFECTION WITH AN OBLIGATE INTRACELLULAR PATHOGEN J. Biol. Chem., January 20, 2006; 281(3): 1652 - 1659. [Abstract] [Full Text] [PDF]
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 S. Appel, V. Mirakaj, A. Bringmann, M. M. Weck, F. Grunebach, and P. Brossart PPAR-{gamma} agonists inhibit toll-like receptor-mediated activation of dendritic cells via the MAP kinase and NF-{kappa}B pathways Blood, December 1, 2005; 106(12): 3888 - 3894. [Abstract] [Full Text] [PDF]
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K. O. Omueti, J. M. Beyer, C. M. Johnson, E. A. Lyle, and R. I. Tapping Domain Exchange between Human Toll-like Receptors 1 and 6 Reveals a Region Required for Lipopeptide Discrimination J. Biol. Chem., November 4, 2005; 280(44): 36616 - 36625. [Abstract] [Full Text] [PDF]
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 C. Feterowski, A. Novotny, S. Kaiser-Moore, P. F. Muhlradt, T. Rossmann-Bloeck, M. Rump, B. Holzmann, and H. Weighardt Attenuated pathogenesis of polymicrobial peritonitis in mice after TLR2 agonist pre-treatment involves ST2 up-regulation Int. Immunol., August 1, 2005; 17(8): 1035 - 1046. [Abstract] [Full Text] [PDF]
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J. F. Hedges, K. J. Lubick, and M. A. Jutila {gamma}{delta} T Cells Respond Directly to Pathogen-Associated Molecular Patterns J. Immunol., May 15, 2005; 174(10): 6045 - 6053. [Abstract] [Full Text] [PDF]
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 H. J. Robertshaw and F. M. Brennan Release of tumour necrosis factor {alpha} (TNF{alpha}) by TNF{alpha} cleaving enzyme (TACE) in response to septic stimuli in vitro Br. J. Anaesth., February 1, 2005; 94(2): 222 - 228. [Abstract] [Full Text] [PDF]
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K.-M. Choe, H. Lee, and K. V. Anderson Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor PNAS, January 25, 2005; 102(4): 1122 - 1126. [Abstract] [Full Text] [PDF]
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E. Kiss-Toth, S. M. Bagstaff, H. Y. Sung, V. Jozsa, C. Dempsey, J. C. Caunt, K. M. Oxley, D. H. Wyllie, T. Polgar, M. Harte, et al. Human Tribbles, a Protein Family Controlling Mitogen-activated Protein Kinase Cascades J. Biol. Chem., October 8, 2004; 279(41): 42703 - 42708. [Abstract] [Full Text] [PDF]
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R. P. Darveau, T.-T. T. Pham, K. Lemley, R. A. Reife, B. W. Bainbridge, S. R. Coats, W. N. Howald, S. S. Way, and A. M. Hajjar Porphyromonas gingivalis Lipopolysaccharide Contains Multiple Lipid A Species That Functionally Interact with Both Toll-Like Receptors 2 and 4 Infect. Immun., September 1, 2004; 72(9): 5041 - 5051. [Abstract] [Full Text] [PDF]
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L. J. Moore, A. C. Pridmore, S. K. Dower, and R. C. Read The glycopeptide vancomycin does not enhance toll-like receptor 2 (TLR2) activation by Streptococcus pneumoniae J. Antimicrob. Chemother., July 1, 2004; 54(1): 76 - 78. [Abstract] [Full Text] [PDF]
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B. P. Leung, D. Xu, S. Culshaw, I. B. McInnes, and F. Y. Liew A Novel Therapy of Murine Collagen-Induced Arthritis with Soluble T1/ST2 J. Immunol., July 1, 2004; 173(1): 145 - 150. [Abstract] [Full Text] [PDF]
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L. C. Parker, M. K. B. Whyte, S. N. Vogel, S. K. Dower, and I. Sabroe Toll-Like Receptor (TLR)2 and TLR4 Agonists Regulate CCR Expression in Human Monocytic Cells J. Immunol., April 15, 2004; 172(8): 4977 - 4986. [Abstract] [Full Text] [PDF]
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 H.-J. Anders, B. Banas, and D. Schlondorff Signaling Danger: Toll-Like Receptors and their Potential Roles in Kidney Disease J. Am. Soc. Nephrol., April 1, 2004; 15(4): 854 - 867. [Abstract] [Full Text] [PDF]
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 D. Zhang, G. Zhang, M. S. Hayden, M. B. Greenblatt, C. Bussey, R. A. Flavell, and S. Ghosh A Toll-like Receptor That Prevents Infection by Uropathogenic Bacteria Science, March 5, 2004; 303(5663): 1522 - 1526. [Abstract] [Full Text] [PDF]
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E. A. Kurt-Jones, M. Chan, S. Zhou, J. Wang, G. Reed, R. Bronson, M. M. Arnold, D. M. Knipe, and R. W. Finberg Herpes simplex virus 1 interaction with Toll-like receptor 2 contributes to lethal encephalitis PNAS, February 3, 2004; 101(5): 1315 - 1320. [Abstract] [Full Text] [PDF]
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L. Guillot, S. Medjane, K. Le-Barillec, V. Balloy, C. Danel, M. Chignard, and M. Si-Tahar Response of Human Pulmonary Epithelial Cells to Lipopolysaccharide Involves Toll-like Receptor 4 (TLR4)-dependent Signaling Pathways: EVIDENCE FOR AN INTRACELLULAR COMPARTMENTALIZATION OF TLR4 J. Biol. Chem., January 23, 2004; 279(4): 2712 - 2718. [Abstract] [Full Text] [PDF]
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P. L. Fisette, S. Ram, J. M. Andersen, W. Guo, and R. R. Ingalls The Lip Lipoprotein from Neisseria gonorrhoeae Stimulates Cytokine Release and NF-{kappa}B Activation in Epithelial Cells in a Toll-like Receptor 2-dependent Manner J. Biol. Chem., November 21, 2003; 278(47): 46252 - 46260. [Abstract] [Full Text] [PDF]
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 K. Peters, R. E. Unger, J. Brunner, and C.J. Kirkpatrick Molecular basis of endothelial dysfunction in sepsis Cardiovasc Res, October 15, 2003; 60(1): 49 - 57. [Abstract] [Full Text] [PDF]
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D. de Kleijn and G. Pasterkamp Toll-like receptors in cardiovascular diseases Cardiovasc Res, October 15, 2003; 60(1): 58 - 67. [Abstract] [Full Text] [PDF]
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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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 F. Sandor, E. Latz, F. Re, L. Mandell, G. Repik, D. T. Golenbock, T. Espevik, E. A. Kurt-Jones, and R. W. Finberg Importance of extra- and intracellular domains of TLR1 and TLR2 in NF{kappa}B signaling J. Cell Biol., September 15, 2003; 162(6): 1099 - 1110. [Abstract] [Full Text] [PDF]
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 E. S. Van Amersfoort, T. J. C. Van Berkel, and J. Kuiper Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock Clin. Microbiol. Rev., July 1, 2003; 16(3): 379 - 414. [Abstract] [Full Text] [PDF]
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A. Yoder, X. Wang, Y. Ma, M. T. Philipp, M. Heilbrun, J. H. Weis, C. J. Kirschning, R. M. Wooten, and J. J. Weis Tripalmitoyl-S-Glyceryl-Cysteine-Dependent OspA Vaccination of Toll-Like Receptor 2-Deficient Mice Results in Effective Protection from Borrelia burgdorferi Challenge Infect. Immun., July 1, 2003; 71(7): 3894 - 3900. [Abstract] [Full Text] [PDF]
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A. C. Pridmore, G. A. Jarvis, C. M. John, D. L. Jack, S. K. Dower, and R. C. Read Activation of Toll-Like Receptor 2 (TLR2) and TLR4/MD2 by Neisseria Is Independent of Capsule and Lipooligosaccharide (LOS) Sialylation but Varies Widely among LOS from Different Strains Infect. Immun., July 1, 2003; 71(7): 3901 - 3908. [Abstract] [Full Text] [PDF]
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G. Melmed, L. S. Thomas, N. Lee, S. Y. Tesfay, K. Lukasek, K. S. Michelsen, Y. Zhou, B. Hu, M. Arditi, and M. T. Abreu Human Intestinal Epithelial Cells Are Broadly Unresponsive to Toll-Like Receptor 2-Dependent Bacterial Ligands: Implications for Host-Microbial Interactions in the Gut J. Immunol., February 1, 2003; 170(3): 1406 - 1415. [Abstract] [Full Text] [PDF]
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M. A. Dobrovolskaia, A. E. Medvedev, K. E. Thomas, N. Cuesta, V. Toshchakov, T. Ren, M. J. Cody, S. M. Michalek, N. R. Rice, and S. N. Vogel Induction of In Vitro Reprogramming by Toll-Like Receptor (TLR)2 and TLR4 Agonists in Murine Macrophages: Effects of TLR "Homotolerance" Versus "Heterotolerance" on NF-{kappa}B Signaling Pathway Components J. Immunol., January 1, 2003; 170(1): 508 - 519. [Abstract] [Full Text] [PDF]
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 K. Takeda, O. Takeuchi, and S. Akira Recognition of lipopeptides by Toll-like receptors Innate Immunity, December 1, 2002; 8(6): 459 - 463. [Abstract] [PDF]
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E. E. Putnins, A.-R. Sanaie, Q. Wu, and J. D. Firth Induction of Keratinocyte Growth Factor 1 Expression by Lipopolysaccharide Is Regulated by CD-14 and Toll-Like Receptors 2 and 4 Infect. Immun., December 1, 2002; 70(12): 6541 - 6548. [Abstract] [Full Text] [PDF]
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H. Weighardt, S. Kaiser-Moore, R. M. Vabulas, C. J. Kirschning, H. Wagner, and B. Holzmann Cutting Edge: Myeloid Differentiation Factor 88 Deficiency Improves Resistance Against Sepsis Caused by Polymicrobial Infection J. Immunol., September 15, 2002; 169(6): 2823 - 2827. [Abstract] [Full Text] [PDF]
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S. E. Applequist, R. P. A. Wallin, and H.-G. Ljunggren Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines Int. Immunol., September 1, 2002; 14(9): 1065 - 1074. [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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O. Takeuchi, S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong, R. L. Modlin, and S. Akira Cutting Edge: Role of Toll-Like Receptor 1 in Mediating Immune Response to Microbial Lipoproteins J. Immunol., July 1, 2002; 169(1): 10 - 14. [Abstract] [Full Text] [PDF]
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I. Sabroe, E. C. Jones, L. R. Usher, M. K. B. Whyte, and S. K. Dower Toll-Like Receptor (TLR)2 and TLR4 in Human Peripheral Blood Granulocytes: A Critical Role for Monocytes in Leukocyte Lipopolysaccharide Responses J. Immunol., May 1, 2002; 168(9): 4701 - 4710. [Abstract] [Full Text] [PDF]
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R. P. Darveau, S. Arbabi, I. Garcia, B. Bainbridge, and R. V. Maier Porphyromonas gingivalis Lipopolysaccharide Is Both Agonist and Antagonist for p38 Mitogen-Activated Protein Kinase Activation Infect. Immun., April 1, 2002; 70(4): 1867 - 1873. [Abstract] [Full Text] [PDF]
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 K. Edfeldt, J. Swedenborg, G. K. Hansson, and Z.-q. Yan Expression of Toll-Like Receptors in Human Atherosclerotic Lesions: A Possible Pathway for Plaque Activation Circulation, March 12, 2002; 105(10): 1158 - 1161. [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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K. A. Zarember and P. J. Godowski Tissue Expression of Human Toll-Like Receptors and Differential Regulation of Toll-Like Receptor mRNAs in Leukocytes in Response to Microbes, Their Products, and Cytokines J. Immunol., January 15, 2002; 168(2): 554 - 561. [Abstract] [Full Text] [PDF]
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A. Fukui, N. Inoue, M. Matsumoto, M. Nomura, K. Yamada, Y. Matsuda, K. Toyoshima, and T. Seya Molecular Cloning and Functional Characterization of Chicken Toll-like Receptors. A SINGLE CHICKEN TOLL COVERS MULTIPLE MOLECULAR PATTERNS J. Biol. Chem., December 7, 2001; 276(50): 47143 - 47149. [Abstract] [Full Text] [PDF]
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A. Mansell, N. Khelef, P. Cossart, and L. A. J. O'Neill Internalin B Activates Nuclear Factor-kappa B via Ras, Phosphoinositide 3-Kinase, and Akt J. Biol. Chem., November 16, 2001; 276(47): 43597 - 43603. [Abstract] [Full Text] [PDF]
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F. Takeshita, C. A. Leifer, I. Gursel, K. J. Ishii, S. Takeshita, M. Gursel, and D. M. Klinman Cutting Edge: Role of Toll-Like Receptor 9 in CpG DNA-Induced Activation of Human Cells J. Immunol., October 1, 2001; 167(7): 3555 - 3558. [Abstract] [Full Text] [PDF]
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M. J. Sweet, B. P. Leung, D. Kang, M. Sogaard, K. Schulz, V. Trajkovic, C. C. Campbell, D. Xu, and F. Y. Liew A Novel Pathway Regulating Lipopolysaccharide-Induced Shock by ST2/T1 Via Inhibition of Toll-Like Receptor 4 Expression J. Immunol., June 1, 2001; 166(11): 6633 - 6639. [Abstract] [Full Text] [PDF]
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M. Hirschfeld, J. J. Weis, V. Toshchakov, C. A. Salkowski, M. J. Cody, D. C. Ward, N. Qureshi, S. M. Michalek, and S. N. Vogel Signaling by Toll-Like Receptor 2 and 4 Agonists Results in Differential Gene Expression in Murine Macrophages Infect. Immun., March 1, 2001; 69(3): 1477 - 1482. [Abstract] [Full Text] [PDF]
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