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*
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037;
Department of Immunology, Saga Medical School, Nabeshima, Saga, Japan; and
Department of Molecular Biology, Genentech, Inc., South San Francisco, CA 94080
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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
(7, 8, 9). Collectively, these findings led to the proposal
that human Toll receptors are evolutionarily conserved components of
the innate immune system. The identification of infectious pathogens
and their products that activate mammalian cells through specific Toll
receptors has been an intense area of research over the past 2
years.
One of the most potent pathogen-derived inflammatory mediators is LPS,
a major structural component of Gram-negative bacteria. Activation of
cells, most notably macrophages, results in activation of NF-B and
production of cytokines and other inflammatory molecules. The
identification of cell surface receptors capable of mediating
LPS-induced inflammatory responses has been an intense area of
investigation for many years (10, 11). The primary
receptor for LPS is CD14, a cell surface marker of macrophages
(12). Although CD14 has been shown to mediate LPS-induced
activation in a wide variety of cell types, this receptor is GPI
anchored and incapable of directly transducing signals across the cell
membrane. Until recently, a receptor that directly transduces an
activation signal in response to LPS has remained elusive.
Shortly after the discovery of mammalian TLRs, transfection of TLR2 was
shown to confer LPS-induced cellular activation of NF-B in human
embryonic kidney 293 cells (13, 14). In this system,
coexpression of CD14 enhances LPS-induced cellular activation mediated
by TLR2. Using transfected human embryonic kidney 293 cells, LPS was
subsequently shown to cause the oligomerization of TLR2 and the
recruitment of IL-1-receptor-associated kinase to the TLR2 complex
(15). In support of a role for TLR2 in LPS signaling,
expression of a dominant-negative form of the TLR2 receptor in RAW
cells was shown to inhibit LPS-induced gene expression driven by the
IL-12 gene promoter. In addition, a mAb directed against TLR2 was shown
to inhibit LPS-mediated IL-12 release from human adherent monocytes
(16). Taken together, this biochemical evidence implicates
TLR2 as a receptor mediating LPS-induced cellular activation.
Concurrent with the studies on TLR2, a point mutation in the signaling
domain of the gene encoding TLR4 was identified as the genetic defect
of the C3H/HeJ mouse, an inbred strain long known to be hyporesponsive
to LPS (17, 18). Subsequent studies revealed that
overexpression of a TLR4 gene harboring this point mutation in RAW
cells results in dampened TNF-
release in response to LPS (19, 20). Targeted disruption of genomic TLR4 in mice has confirmed
that the TLR4 receptor is necessary for sensitive responses to LPS
(21). In addition, certain cell lines transfected with
human TLR4 acquire the ability to activate NF-B in response to LPS
(22, 23). Thus, genetic and biochemical evidence has
identified TLR4 as a receptor that mediates cellular activation in
response to LPS.
The idea of whether TLR2 or TLR4 represents the physiologically
relevant LPS receptor has been the source of some debate especially as
recently generated TLR2 knockout mice appear to have no significant
defects in LPS responsiveness (24). In this study, we have
studied the contribution of both TLR2 and TLR4 to LPS-induced
production of TNF- and IL-8 in both human cell lines and human whole
blood. Through transfection studies and the use of blocking monoclonal
anti-TLR2 and anti-TLR4 Abs, we have found that the
contribution of each TLR to LPS-induced inflammatory responses is
dependent upon both the cell type used and the source of the LPS. Most
importantly, we have observed that after repurification, commercial
preparations of both Escherichia coli and Salmonella
minnesota LPS no longer induce cellular activation through TLR2,
indicating that impurities in these LPS preparations are responsible
for the observed TLR2-mediated signaling. Our results show that TLR4,
and not TLR2, is the predominant receptor mediating LPS-induced
cellular activation in human whole blood.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heat-killed Staphylococcus aureus (HKSA) was a kind gift of Dr. Colleen Fearns (The Scripps Research Institute, La Jolla, CA). Soluble peptidoglycan (PGN) purified from S. aureus was a kind gift of Dr. Roman Dziarski (Indiana University School of Medicine, Gary, IN). All commercial LPS preparations were purchased from List Biological Laboratories (Cambell, CA). Polymyxin B was obtained from Sigma (St. Louis, MO). The anti-CD14 mAb 63D3 was obtained from the American Type Culture Collection (Manassas, VA). The anti-CD14 mAb 28C5 was a gift from Dr. A. Moriarty and Dr. D. Leturcq (R.W. Johnson Pharmaceutical Research Institute, La Jolla, CA). The anti-TLR2 mAbs mAb 2380 and mAb 2392 have been previously described (25). The mAbs HTA405, HTA414, and HTA1216, against human TLR4, were generated by immunizing mice with TLR4-expressing Ba/F3 cells (23). Spleen cells from immunized mice were fused with SP2/0 myeloma cells and hybridomas were chosen that specifically stain TLR4-expressing Ba/F3 cells as described (23).
Repurification of LPS
Commercial LPS preparations were resuspended in endotoxin-free water containing 0.2% triethylamine followed by vortexing. A portion of each commercial LPS preparation was repurified using a modified phenol-water extraction procedure followed by ethanol precipitation as described (26, 27, 28). Recovery of LPS was determined by colitose assay for smooth LPS types and 3-deoxy-D-manno-octulosonic acid assay for rough LPS types as described (29). LPS recoveries were typically 65–85% and were confirmed by performing a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) according to a protocol supplied by the manufacturer.
Construction of TLR4 expressing THP-1 cell lines
The cDNA encoding full length TLR4 was amplified by PCR using the primers 5'-CCTTGGTACCAAGCTTCTCGAGGCCAGGATGATGTCTGCCTCG-3' and 5'-CCTTGGATCCGTCGACCTATCAGATAGATGTTGCTTCCTGCC-3'. The resulting amplified DNA was cloned as a XhoI/BamHI fragment into the XhoI and BglII sites of the retroviral vector pMSCV-blasto (Gary Nolan, Stanford University School of Medicine, Stanford, CA) to create the plasmid pMSCV-blasto-TLR4. This construct was confirmed by sequencing at the Core Facility of The Scripps Research Institute. Pheonix-Ampho cells, an amphotropic retroviral packing cell line derived from 293T cells (30, 31), were transfected with pMSCV-blasto-TLR4 by the calcium phosphate method. Replication-defective retrovirus was harvested from the cell supernatant 48 h after transfection and sterile filtered. The macrophage cell line THP1-CD14, a monocytic cell line that constitutively expresses CD14 (32, 33), was infected by centrifuging the cells in medium containing retrovirus plus 5 µg/ml polybrene (30, 31). The medium was changed 24 h postinfection and cells were selected as a batch in medium containing 5 µg/ml blasticidin. Vector control THP1-CD14 cells were generated by following the above procedure using the empty vector pMSCV-blasto. Cells containing stably integrated vector sequences were selected for 3 wk in medium containing 5 µg/ml blasticidin.
Cell activation assays
THP1-CD14 cells were grown in RPMI 1640 medium containing 10% FBS, 1% penicillin/streptomycin/glutamine, and 800 µg/ml G418, to maintain CD14 expression. On the day of the assay, the cells were washed three times in serum-free RPMI 1640 medium and resuspended in the same medium containing 2% human serum. About 105 cells were added to each well of a 96-well plate and activated with various agonists as indicated in the figures. Upon the addition of an agonist, the cells were placed in an incubator at 37°C and 5% carbon dioxide for 6 h. Where indicated, Abs were preincubated with cells for 30 min at a concentration of 10 µg/ml before agonist addition.
U373 cells, a human astrocytoma cell line, were grown in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin/glutamine. The day before the assay, the cells were trypsinized, transferred into 96-well plates, and allowed to recover overnight. The adherent cells were washed three times with serum-free RPMI 1640 medium. Cells were stimulated for 6 h in the presence of 2% human serum using Re595 or 0111B4 LPS as indicated in the figure legends. The amount of LPS used in the Ab blocking experiments reflects the fact that the average molecular mass of rough Re595 LPS is 10-fold less than that of smooth 0111B4 LPS. Where indicated, Abs were preincubated with cells for 30 min at a concentration of 10 µg/ml before agonist addition.
PCR analysis
Total RNA was isolated from U373 cells using TRIzol reagent (Life Technologies, Rockville, MD) according to protocols supplied by the manufacturer. The synthesis of cDNA was performed, in the presence and absence of reverse transcriptase, using the Superscript Preamplification system (Life Technologies) and oligo(dT) as recommended by the manufacturer. PCR amplification of 500 ng cDNA was performed using Taq polymerase (Life Technologies) and TLR2- or TLR4-specific primers. The sequence of the primers for TLR2 were 5'-GGCCAGCAAATTACCTGTGT-3' and 5'-TTCTCCACCCAGTAGGCATC-3'. The sequences of the primers for TLR4 were 5'-TGAGCAGTCGTGCTGGTATC-3' and 5'-CAGGGCTTTTCTGAGTCGTC-3'. PCR products were analyzed on gels composed of 2% NuSieve (FMC, Chicago, IL) and 1% agarose and the DNA was visualized with ethidium bromide.
Northern blot analysis
Total RNA was isolated from either THP-1 cells or HUVECs using TRIzol reagent (Life Technologies) according to protocols supplied by the manufacturer. RNA samples, 10 µg per lane, were separated on 1% agarose-formaldehyde gels and transferred to nitrocellulose (BA85, Schleicher & Schuell, Keene, NH) in 20x SSC overnight (1x SSC is 0.15 M NaCl and 0.015 M sodium citrate). The membranes were prehybridized for 6 h at 60°C in hybridization buffer (50 mM PIPES/50 mM sodium phosphate (pH 7), 100 mM NaCl, 0.1 mM EDTA, 5% SDS containing 200 µg/ml yeast RNA and 50 µg/ml salmon sperm DNA) then hybridized overnight in the same buffer containing 32P-labeled DNA probes for either TLR2, TLR4, or GAPDH. The filter was washed with 1x SSC followed by autoradiography. The probe for TLR2 was generated by random priming of the 1002-bp NdeI to EcoRI fragment of the TLR2 gene. The probe for TLR4 was generated by random priming of the 621-bp PstI to EcoRI fragment of the TLR4 gene. The probe for GAPDH was generated by hybridizing the two partially complementary oligonucleotides for GAPDH, 5'-ATGTTCCAGTATGATTCCACCCACGGCAAGTTCCACGGCACGGTCAAGGCTGAGAACGGG-3' and 5'-ATCTCGCTCCTGGAAGATGGTGATGGCCTTCCCGTTGATGACCAGCTTCCCGTTCTCAGC-3', followed by labeling using the Klenow fragment of DNA polymerase I.
Flow cytometry analysis
THP1-CD14 cells were washed twice in ice cold FACS buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 1% BSA). Approximately 105 cells were incubated for 1 h on ice in FACS buffer containing 5% normal rabbit serum and 5 µg/ml primary Ab. The cells were washed and labeled in FACS buffer containing 5% normal rabbit serum and FITC sheep anti-mouse IgG (PharMingen, La Jolla, CA). After a final wash, the cells were resuspended in FACS buffer and analyzed by flow cytometry using a FACScan flow cytometer (Becton Dickinson).
Whole blood activation assay
Whole blood from healthy donors was collected into tubes containing heparin the day of the assay. The blood was diluted 1:4 in RPMI 1640 medium and aliquoted into 96-well plates. Upon the addition of agonist, the plate was placed in an incubator at 37°C and 5% carbon dioxide for 4 h. Where indicated, Abs were preincubated in blood for 30 min at a concentration of 10 µg/ml before agonist addition.
ELISAs
After incubation, cell supernatants were removed and assayed for
cytokine production by standard sandwich ELISA using 96-well Immunlon
plates (Dynatech Laboratories, Chantilly, VA). The TNF- ELISA was
performed using mAbs 68B6A3 or 68B2B3 for capture and the biotinylated
mAb 68B3C5 (BioSource International, Camarillo, CA) followed by
streptavidin HRP for detection. The IL-8 ELISA was performed using the
mAb MAB208 for capture and a biotinylated polyclonal rabbit
anti-human IL-8 Ab (R&D Systems, Minneapolis, MN) followed by
streptavidin HRP for detection. The IL-6 ELISA was performed using
polyclonal goat anti-human IL-6 (R&D systems, Minneapolis, MN) for
capture and polyclonal rabbit anti-human IL-6 (Endogen, Woburn, MA)
followed by HRP-conjugated goat-anti-rabbit IgG for detection
(BioSource International). All ELISAs were developed using
o-phenylenediamine as a substrate, and OD was determined at
a wavelength of 490 nm using a Spectramax plate reader and software
(Molecular Devices, Sunnyvale, CA). All values were interpolated from
either a log-log or a four-parameter fit of a curve generated from
appropriate standards.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-8. Overexpression of TLR4
had no effect on the magnitude or sensitivity of THP1-CD14 cells in
response to PMA, HKSA, or PGN (Fig. 1
).
In contrast, THP1-CD14 cells that overexpress TLR4 exhibited increased
sensitivity and responsiveness to both Re595 LPS and 0111B4 LPS. These
effects on cellular activation were reflected in measurements of both
TNF- and IL-8 release. Taken together, these results show that TLR4
enhances the ability of THP1-CD14 monocytic cells to respond to LPS. It
is noteworthy that the magnitude of the cytokine response of THP1-CD14
cells to 0111B4 LPS was at least 20-fold higher than the response to
Re595 LPS.
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, none of the aforementioned Abs had any
effect on PMA-mediated activation of THP1-CD14 cells as measured by
TNF- or IL-8 production. In addition, none of the anti-TLR4 Abs
had any measurable effect on HKSA- or PGN-mediated cytokine release.
However, cellular activation induced by either HKSA or PGN was almost
completely blocked by mAb 2392, an antagonistic anti-TLR2
Ab.
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, cytokine release of THP1-CD14 cells in response to
Re595 LPS was at least 20-fold weaker than to 0111B4 LPS. Polymyxin B,
a cationic peptide that inhibits LPS-induced cellular activation,
blocked at least 80% of both Re595- and 0111B4-mediated cytokine
release. In addition, 28C5, an Ab that blocks binding of LPS to CD14,
greatly inhibited cellular responses to both Re595 LPS and 0111B4 LPS.
Taken together, these results indicate that TLR2 and TLR4 are the
predominant receptors of THP1-CD14 cells that mediate activation in
response to 0111B4 LPS and Re595 LPS, respectively. Thus, the relative
contribution of TLR2 and TLR4 to LPS-induced monocyte activation is
dependent upon the type or source of LPS used.
To ascertain the generality of the above results we also tested the
effects of the anti-TLR2 and anti-TLR4 Abs on the LPS-induced
activation of U373 cells. U373 cells are a cell line derived from a
human astrocytoma and release IL-6 in response to LPS in a
CD14-dependent fashion (37). In contrast to THP1-CD14
cells, Re595 LPS and 0111B4 LPS induced roughly comparable levels of
cytokine release in U373 cells (Fig. 3B). We also observed that the
anti-TLR4 Ab HTA405 inhibited 
50 and 20% of IL-6 release from
U373 cells induced with Re595 LPS and 0111B4 LPS, respectively (Fig. 3C). As expected, activation mediated by either Re595 or
0111B4 LPS was almost completely abrogated both by the anti-CD14 Ab
28C5 and by polymyxin B. Surprisingly, the anti-TLR2 Abs had no
effect on cellular activation mediated by 0111B4 LPS indicating that,
in contrast to THP1-CD14 cells, TLR2 makes no contribution to this
event in U373 cells. This observation prompted us examine the
expression of TLR2 and TLR4 in U373 cells. RT-PCR analysis clearly
detected the presence of TLR4 message but did not detect any TLR2
message in U373 cells (Fig. 3A). This finding has been
previously noted (38), and demonstrates that U373 cells do
not express TLR2. In support of this, we have found that neither HKSA
or PGN activate IL-6 release of U373 cells (data not shown). Taken
together, these results demonstrate that cellular responses to LPS
occur in the absence of TLR2 and that the contribution of TLR2 and TLR4
to LPS-induced cellular activation is also a function of cell
type.
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). RT-PCR analysis has shown that THP-1 cells possess
message for both TLR2 and TLR4 (39). A quantitative
northern blot analysis revealed that, in sharp contrast to HUVECs,
THP-1 cells possess high levels of TLR2 message but barely detectable
levels of TLR4 message (Fig. 4A). In support of this, FACS
analysis indicates that cell surface expression of TLR2 is much higher
than that of TLR4 in THP1-CD14 cells (Fig. 4B). Taken
together with the data obtained using U373 cells, these results suggest
that the cell-type-specific contribution of TLR2 and TLR4 to 0111B4
LPS-induced cellular activation may simply reflect different TLR
expression levels. In support of this we have shown that overexpression
of TLR4 in THP1-CD14 cells renders them more responsive to LPS (Fig. 1)
and this enhanced response is inhibited by anti-TLR4 Abs (data not
shown). Despite the significant expression level of TLR2 and the strong
contribution of TLR2 to cytokine release induced by HKSA, PGN, or
0111B4 LPS, there is no measurable contribution of this receptor to
cytokine release induced by Re595 LPS.
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). In general, we found that weakly
activating LPSs were significantly inhibited by anti-TLR4 Abs and
that strongly activating LPSs were greatly inhibited by the
anti-TLR2 Ab (Fig. 5). Moreover, we found that the level of
stimulation, or the effect of anti-TLR Abs on LPS activities, could
not be predicted based on the known structure of the LPSs or the
bacterial species from which they were derived.
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). Moreover,
while the anti-TLR4 Abs inhibited at least 50% of the TNF release
induced by every repurified LPS, the anti-TLR2 Ab had no effect on
the activity of any repurified LPS. Similar results were obtained using
repurified LCD25 and K235 LPS from E. coli as well as
repurified Rb, Rc, and Rd LPS from S. minnesota (data not
shown). Taken together, these results demonstrate that upon
repurification commercial LPS preparations lose the ability to mediate
cellular activation through TLR2 suggesting that impurities in these
LPS preparations are responsible for the TLR2-mediated signaling. In
contrast, repurified LPS appears to retain the ability to activate
cells through TLR4 indicating that this receptor is a signaling
receptor for E. coli and S. minnesota
LPS.
To ascertain the contribution of TLR2 and TLR4 to LPS-induced immune
responses under more physiologic conditions, we tested the effect of
the anti-TLR2 and anti-TLR4 Abs on LPS-mediated cytokine
release in human whole blood. Fig. 6
shows that all the anti-TLR4 Abs significantly inhibited TNF-
and IL-8 release from whole blood treated with either commercial Re595
or 0111B4 LPS. In contrast, the anti-TLR2 Abs had no effect on
cytokine production in whole blood treated with Re595 LPS and only
marginally, but reproducibly, inhibited cytokine production induced by
commercial 0111B4 LPS. This low level of inhibition was only observed
at higher LPS concentrations in agreement with the idea
that the TLR2-mediated response is due to impurities in the commercial
0111B4 LPS. The anti-TLR2 Ab mAb 2392 specifically blocked cytokine
release induced by PGN, demonstrating that this Ab is blocking the TLR2
receptor under the conditions of this whole blood assay. As expected,
the anti-CD14 mAb 28C5 dramatically inhibited LPS-induced cytokine
production in whole blood.
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). The anti-TLR2 Ab only
weakly inhibited whole blood TNF release mediated by commercial LPS
preparations and this weak inhibition was not detected using repurified
LPSs. Taken together, these results confirm that impurities in
commercial LPS preparations activate cells through TLR2. In addition,
these results demonstrate that in human whole blood, TLR4 is a
predominant receptor mediating LPS-induced cellular activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To determine a structural basis for the difference in activity observed between 0111B4 and Re595 LPS we extended our experiments to a range of rough and smooth LPS types from both E. coli and S. minnesota. However, the differences in cellular activity we observed that were mediated by TLR2 could not be ascribed to the known different structural features between these LPSs. In this regard, LPS has proven difficult to completely purify and there exists a long history of contaminating molecules in LPS with cell stimulatory activity (reviewed in Refs. 26, 27). Therefore, we repurified the commercial LPS preparations using a modified phenol-water extraction method followed by ethanol precipitation, a procedure that has been shown to remove trace proteins (26, 27). Repurification of the commercial LPS preparations eliminated TLR2-mediated cell signaling but retained TLR4-mediated cell signaling, demonstrating that TLR4, but not TLR2, is a signaling receptor for pure E. coli- and S. minnesota-derived LPS.
The idea that TLR4, and not TLR2, mediates LPS-induced cellular
activation has been strongly argued in a recent review
(44). Central to this argument is that a mutation in the
gene encoding TLR4 underlies the genetic defect of C3H/HeJ mice, an
inbred strain that is hyporesponsive to LPS (17, 18).
Accordingly, targeted disruption of the TLR4 gene in mice results in an
LPS-hyporesponsive phenotype (21). In this regard, it is
noteworthy that C3H/HeJ macrophages exposed to IFN-
, a treatment
that is thought to render them sensitive to LPS, are not responsive to
LPS preparations repurified by the modified phenol-water extraction
method used in this study (26, 41, 42). Thus, that IFN-
reverses the sensitivity of C3H/HeJ macrophages to LPS
(45) is likely attributable to contaminants within the LPS
preparations.
Lipid A has long been established as the bioactive component of LPS
(reviewed in Ref. 46). As this is the common structural
feature shared by all of the LPSs we tested, it is reasonable that upon
repurification all of our LPSs exhibit similar levels of biological
activity, mediated through TLR4, in our assays. The idea that TLR4 is
responsive to the lipid A portion of LPS gains support from the finding
that this receptor appears to mediate the species specific responses
induced by synthetic lipid IVa, a precursor and closely related
structure to lipid A (47, 48). Additionally, several lines
of evidence have shown that TLR2 is not required for mediating
LPS-induced signaling. One group has found that hamster cells
genomically encode a nonfunctional gene for TLR2 even though these
cells are fully LPS responsive (49). In agreement with
this, U373 cells also retain LPS responsiveness despite a lack of
expression of TLR2 (Fig. 3). Most compelling is the observation that
recently generated TLR2 knockout mice have no significant defects in
LPS responsiveness (24). Together, these findings
demonstrate that TLR2 is not required for LPS signaling and suggest
that this receptor plays no significant physiologic role in LPS
signaling. Interestingly, LPSs derived from oral black-pigmented
bacteria, Porphyromonas gingivalis and Prevotella
intermedia, whose lipid A structures are different from the
commonly studied LPSs of Enterobacteriaceae, stimulate
C3H/HeJ macrophages even after repurification by the method described
in this study (50). Thus, the idea that TLR4 is a
signaling receptor for lipid A may be restricted to LPSs derived from
the family of Enterobacteriaceae.
The idea that impurities in LPS are responsible for TLR2-mediated signaling is supported by several observations in the literature. TLR2 was initially identified as an LPS receptor by stimulating cells that over express TLR2 with commercial LPS preparations (13, 14), an approach confirmed by a number of studies (16, 25, 38, 51, 52, 53). Accordingly, we have found that THP1-CD14 cells that express significantly greater levels of TLR2 compared with TLR4 are most sensitive to impure commercial LPS preparations. The contention that the observed activation is due to impurities in the LPS is supported by studies showing that the sensitivity of TLR2 transfected cells to other TLR2 agonists is severalfold greater than it is to LPS (25, 38). In addition, in contrast to the robust effect of TLR4, overexpression of TLR2 has been shown to only modestly enhance LPS responsiveness of Chinese hamster ovary cells (52). Moreover, during our studies we became aware of results demonstrating that repurification of LPS by the modified phenol-water extraction method used in this study eliminates activation of cells transfected with TLR2, but maintains activation of cells transfected with TLR4 (28). Our data are a direct extension of these studies and have confirmed the original observation that was made with E. coli smooth and rough LPS preparations. Moreover, we have now demonstrated this to be true of S. minnesota LPS and have found TLR4 to be the predominant signaling molecule in human peripheral blood cells stimulated with repurified LPS preparations.
At present, we do not know the nature of the contaminant(s) in commercial LPS preparations responsible for TLR2-dependent signaling. Recent studies have implicated TLR2 as a mediator of cellular responses to a wide variety of infectious pathogens and their products including yeast cell walls (19), spirochetal lipoproteins (16, 38, 54), whole mycobacteria (55), mycobacterial lipoarabinomannan (52, 56), whole Gram-positive bacteria (19, 57), as well as Gram-positive bacterial lipotechoic acid (51)and PGN (24, 51, 54, 57). The fact that our TLR2-dependent signaling is markedly inhibited by an anti-CD14 Ab demonstrates that this signaling is also mediated through CD14. In this regard, CD14, like TLR2, has been implicated as a receptor for a variety of fungal and bacterial products (58, 59, 60, 61). The TLR2-dependent activation induced by commercial LPS preparations is inhibited by polymyxin B, a cationic peptide that has historically been used to block LPS-induced cellular stimulation. At present we do not know whether polymyxin B is inhibiting TLR2-dependent cellular activation by interacting with the impurity itself or by interacting with the LPS with which it is associated. Regardless, these results demonstrate a necessity to carefully assess the purity and potency of pathogen-derived components when assigning their actions to the function of individual TLRs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter S. Tobias, Department of Immunology, IMM-12, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.
3 Abbreviations used in this paper: TLR, Toll-like receptor; HKSA, heat-killed Staphylococcus aureus; PGN, peptidoglycan.
Received for publication January 14, 2000. Accepted for publication August 28, 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.B through interleukin-1 signaling mediators in cultured human dermal endothelial cells and human mononuclear phagocytes. J. Biol. Chem. 274:7611.-primed, C3H/HeJ macrophages to a fully tumoricidal state. J. Immunol. 139:3697.[Abstract]
-primed C3H/HeJ (Lpsd) macrophages requires the presence of lipid A-associated proteins. J. Immunol. 131:4196.
interferon on cachectin expression by mononuclear phagocytes: reversal of the lps d (endotoxin resistance) phenotype. J. Exp. Med. 164:1791.
and tumor necrosis factor- by Mycobacterium tuberculosis components. J. Clin. Invest. 91:2076.
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H. S. Seo, S. M. Michalek, and M. H. Nahm Lipoteichoic Acid Is Important in Innate Immune Responses to Gram-Positive Bacteria Infect. Immun., January 1, 2008; 76(1): 206 - 213. [Abstract] [Full Text] [PDF]
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V. Souvannavong, N. Saidji, and R. Chaby Lipopolysaccharide from Salmonella enterica Activates NF-{kappa}B through both Classical and Alternative Pathways in Primary B Lymphocytes Infect. Immun., October 1, 2007; 75(10): 4998 - 5003. [Abstract] [Full Text] [PDF]
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Y. J. Kang, B. Kusler, M. Otsuka, M. Hughes, N. Suzuki, S. Suzuki, W.-C. Yeh, S. Akira, J. Han, and P. P. Jones Calcineurin Negatively Regulates TLR-Mediated Activation Pathways J. Immunol., October 1, 2007; 179(7): 4598 - 4607. [Abstract] [Full Text] [PDF]
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N. Foster, S. R. Lea, P. M. Preshaw, and J. J. Taylor Pivotal Advance: Vasoactive intestinal peptide inhibits up-regulation of human monocyte TLR2 and TLR4 by LPS and differentiation of monocytes to macrophages J. Leukoc. Biol., April 1, 2007; 81(4): 893 - 903. [Abstract] [Full Text] [PDF]
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T. Into, Y. Kanno, J.-i. Dohkan, M. Nakashima, M. Inomata, K.-i. Shibata, C. J. Lowenstein, and K. Matsushita Pathogen Recognition by Toll-like Receptor 2 Activates Weibel-Palade Body Exocytosis in Human Aortic Endothelial Cells J. Biol. Chem., March 16, 2007; 282(11): 8134 - 8141. [Abstract] [Full Text] [PDF]
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J. S. Duffield, S. Hong, V. S. Vaidya, Y. Lu, G. Fredman, C. N. Serhan, and J. V. Bonventre Resolvin D Series and Protectin D1 Mitigate Acute Kidney Injury J. Immunol., November 1, 2006; 177(9): 5902 - 5911. [Abstract] [Full Text] [PDF]
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J. M. Buckley, J. H. Wang, and H. P. Redmond Cellular reprogramming by gram-positive bacterial components: a review J. Leukoc. Biol., October 1, 2006; 80(4): 731 - 741. [Abstract] [Full Text] [PDF]
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J. M. Marques, R. J. Rodrigues, A. C. de Magalhaes-Sant'Ana, and T. Goncalves Saccharomyces cerevisiae Hog1 Protein Phosphorylation upon Exposure to Bacterial Endotoxin J. Biol. Chem., August 25, 2006; 281(34): 24687 - 24694. [Abstract] [Full Text] [PDF]
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N. Feng, M. C. Jaimes, N. H. Lazarus, D. Monak, C. Zhang, E. C. Butcher, and H. B. Greenberg Redundant Role of Chemokines CCL25/TECK and CCL28/MEC in IgA+ Plasmablast Recruitment to the Intestinal Lamina Propria After Rotavirus Infection J. Immunol., May 15, 2006; 176(10): 5749 - 5759. [Abstract] [Full Text] [PDF]
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G. Gatti, V. Rivero, R. D. Motrich, and M. Maccioni Prostate epithelial cells can act as early sensors of infection by up-regulating TLR4 expression and proinflammatory mediators upon LPS stimulation J. Leukoc. Biol., May 1, 2006; 79(5): 989 - 998. [Abstract] [Full Text] [PDF]
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J. S. Yang, H. J. Kim, Y. H. Ryu, C.-H. Yun, D. K. Chung, and S. H. Han Endotoxin contamination in commercially available pokeweed mitogen contributes to the activation of murine macrophages and human dendritic cell maturation. Clin. Vaccine Immunol., March 1, 2006; 13(3): 309 - 313. [Abstract] [Full Text] [PDF]
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P. W. Askenase, A. Itakura, M. C. Leite-de-Moraes, M. Lisbonne, S. Roongapinun, D. R. Goldstein, and M. Szczepanik TLR-Dependent IL-4 Production by Invariant V{alpha}14+J{alpha}18+ NKT Cells to Initiate Contact Sensitivity In Vivo J. Immunol., November 15, 2005; 175(10): 6390 - 6401. [Abstract] [Full Text] [PDF]
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S. C. Gangloff, U. Zahringer, C. Blondin, M. Guenounou, J. Silver, and S. M. Goyert Influence of CD14 on Ligand Interactions between Lipopolysaccharide and Its Receptor Complex J. Immunol., September 15, 2005; 175(6): 3940 - 3945. [Abstract] [Full Text] [PDF]
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G. Mancuso, A. Midiri, C. Biondo, C. Beninati, M. Gambuzza, D. Macri, A. Bellantoni, A. Weintraub, T. Espevik, and G. Teti Bacteroides fragilis-Derived Lipopolysaccharide Produces Cell Activation and Lethal Toxicity via Toll-Like Receptor 4 Infect. Immun., September 1, 2005; 73(9): 5620 - 5627. [Abstract] [Full Text] [PDF]
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 X. Yang, D. Coriolan, V. Murthy, K. Schultz, D. T. Golenbock, and D. Beasley Proinflammatory phenotype of vascular smooth muscle cells: role of efficient Toll-like receptor 4 signaling Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1069 - H1076. [Abstract] [Full Text] [PDF]
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 A. A. Steiner, S. Chakravarty, J. R. Robbins, A. S. Dragic, J. Pan, M. Herkenham, and A. A. Romanovsky Thermoregulatory responses of rats to conventional preparations of lipopolysaccharide are caused by lipopolysaccharide per se-- not by lipoprotein contaminants Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2005; 289(2): R348 - R352. [Abstract] [Full Text] [PDF]
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 D.R. Dixon and R.P. Darveau Lipopolysaccharide Heterogeneity: Innate Host Responses to Bacterial Modification of Lipid A Structure Journal of Dental Research, July 1, 2005; 84(7): 584 - 595. [Abstract] [Full Text] [PDF]
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E. A. Hayashi, S. Akira, and A. Nobrega Role of TLR in B Cell Development: Signaling through TLR4 Promotes B Cell Maturation and Is Inhibited by TLR2 J. Immunol., June 1, 2005; 174(11): 6639 - 6647. [Abstract] [Full Text] [PDF]
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G. C. O'Brien, J. H. Wang, and H. P. Redmond Bacterial Lipoprotein Induces Resistance to Gram-Negative Sepsis in TLR4-Deficient Mice via Enhanced Bacterial Clearance J. Immunol., January 15, 2005; 174(2): 1020 - 1026. [Abstract] [Full Text] [PDF]
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D. S. Zamboni, M. A. Campos, A. C. T. Torrecilhas, K. Kiss, J. E. Samuel, D. T. Golenbock, F. N. Lauw, C. R. Roy, I. C. Almeida, and R. T. Gazzinelli Stimulation of Toll-like Receptor 2 by Coxiella burnetii Is Required for Macrophage Production of Pro-inflammatory Cytokines and Resistance to Infection J. Biol. Chem., December 24, 2004; 279(52): 54405 - 54415. [Abstract] [Full Text] [PDF]
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C. Erridge, A. Pridmore, A. Eley, J. Stewart, and I. R. Poxton Lipopolysaccharides of Bacteroides fragilis, Chlamydia trachomatis and Pseudomonas aeruginosa signal via Toll-like receptor 2 J. Med. Microbiol., August 1, 2004; 53(8): 735 - 740. [Abstract] [Full Text] [PDF]
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C. von Meyenburg, B. H. Hrupka, D. Arsenijevic, G. J. Schwartz, R. Landmann, and W. Langhans Role for CD14, TLR2, and TLR4 in bacterial product-induced anorexia Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2004; 287(2): R298 - R305. [Abstract] [Full Text] [PDF]
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F. Wong, C. Hull, R. Zhande, J. Law, and A. Karsan Lipopolysaccharide initiates a TRAF6-mediated endothelial survival signal Blood, June 15, 2004; 103(12): 4520 - 4526. [Abstract] [Full Text] [PDF]
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N. L. S. Que-Gewirth, A. A. Ribeiro, S. R. Kalb, R. J. Cotter, D. M. Bulach, B. Adler, I. S. Girons, C. Werts, and C. R. H. Raetz A Methylated Phosphate Group and Four Amide-linked Acyl Chains in Leptospira interrogans Lipid A: THE MEMBRANE ANCHOR OF AN UNUSUAL LIPOPOLYSACCHARIDE THAT ACTIVATES TLR2 J. Biol. Chem., June 11, 2004; 279(24): 25420 - 25429. [Abstract] [Full Text] [PDF]
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J. Branger, S. Knapp, S. Weijer, J. C. Leemans, J. M. Pater, P. Speelman, S. Florquin, and T. van der Poll Role of Toll-Like Receptor 4 in Gram-Positive and Gram-Negative Pneumonia in Mice Infect. Immun., February 1, 2004; 72(2): 788 - 794. [Abstract] [Full Text] [PDF]
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 M. A. Elovitz, Z. Wang, E. K. Chien, D. F. Rychlik, and M. Phillippe A New Model for Inflammation-Induced Preterm Birth: The Role of Platelet-Activating Factor and Toll-Like Receptor-4 Am. J. Pathol., November 1, 2003; 163(5): 2103 - 2111. [Abstract] [Full Text] [PDF]
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J. Watanabe, Y. Miyazaki, G. A. Zimmerman, K. H. Albertine, and T. M. McIntyre Endotoxin Contamination of Ovalbumin Suppresses Murine Immunologic Responses and Development of Airway Hyper-reactivity J. Biol. Chem., October 24, 2003; 278(43): 42361 - 42368. [Abstract] [Full Text] [PDF]
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M. Triantafilou and K. Triantafilou Receptor cluster formation during activation by bacterial products Innate Immunity, October 1, 2003; 9(5): 331 - 335. [Abstract] [PDF]
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S. H. Han, J. H. Kim, M. Martin, S. M. Michalek, and M. H. Nahm Pneumococcal Lipoteichoic Acid (LTA) Is Not as Potent as Staphylococcal LTA in Stimulating Toll-Like Receptor 2 Infect. Immun., October 1, 2003; 71(10): 5541 - 5548. [Abstract] [Full Text] [PDF]
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 S. Nakahara, T. Tsunoda, T. Baba, S. Asabe, and H. Tahara Dendritic Cells Stimulated with a Bacterial Product, OK-432, Efficiently Induce Cytotoxic T Lymphocytes Specific to Tumor Rejection Peptide Cancer Res., July 15, 2003; 63(14): 4112 - 4118. [Abstract] [Full Text] [PDF]
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M. Martin, R. E. Schifferle, N. Cuesta, S. N. Vogel, J. Katz, and S. M. Michalek Role of the Phosphatidylinositol 3 Kinase-Akt Pathway in the Regulation of IL-10 and IL-12 by Porphyromonas gingivalis Lipopolysaccharide J. Immunol., July 15, 2003; 171(2): 717 - 725. [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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B. Gao and M.-F. Tsan Recombinant Human Heat Shock Protein 60 Does Not Induce the Release of Tumor Necrosis Factor {alpha} from Murine Macrophages J. Biol. Chem., June 13, 2003; 278(25): 22523 - 22529. [Abstract] [Full Text] [PDF]
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M. Muroi, T. Ohnishi, S. Azumi-Mayuzumi, and K.-i. Tanamoto Lipopolysaccharide-Mimetic Activities of a Toll-Like Receptor 2-Stimulatory Substance(s) in Enterobacterial Lipopolysaccharide Preparations Infect. Immun., June 1, 2003; 71(6): 3221 - 3226. [Abstract] [Full Text] [PDF]
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M. Martin, S. M. Michalek, and J. Katz Role of Innate Immune Factors in the Adjuvant Activity of Monophosphoryl Lipid A Infect. Immun., May 1, 2003; 71(5): 2498 - 2507. [Abstract] [Full Text] [PDF]
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T. Morichika, H. K. Takahashi, H. Iwagaki, T. Yoshino, R. Tamura, M. Yokoyama, S. Mori, T. Akagi, M. Nishibori, and N. Tanaka Histamine Inhibits Lipopolysaccharide-Induced Tumor Necrosis Factor-{alpha} Production in an Intercellular Adhesion Molecule-1- and B7.1-Dependent Manner J. Pharmacol. Exp. Ther., February 1, 2003; 304(2): 624 - 633. [Abstract] [Full Text] [PDF]
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J. H. Wang, B. J. Manning, Q. D. Wu, S. Blankson, D. Bouchier-Hayes, and H. P. Redmond Endotoxin/Lipopolysaccharide Activates NF-{kappa}B and Enhances Tumor Cell Adhesion and Invasion Through a {beta}1 Integrin-Dependent Mechanism J. Immunol., January 15, 2003; 170(2): 795 - 804. [Abstract] [Full Text] [PDF]
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J. H. Wang, M. Doyle, B. J. Manning, S. Blankson, Q. D. Wu, C. Power, R. Cahill, and H. P. Redmond Cutting Edge: Bacterial Lipoprotein Induces Endotoxin-Independent Tolerance to Septic Shock J. Immunol., January 1, 2003; 170(1): 14 - 18. [Abstract] [Full Text] [PDF]
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H. Karlsson, C. Hessle, and A. Rudin Innate Immune Responses of Human Neonatal Cells to Bacteria from the Normal Gastrointestinal Flora Infect. Immun., December 1, 2002; 70(12): 6688 - 6696. [Abstract] [Full Text] [PDF]
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 F M Ruemmele, J F Beaulieu, S Dionne, E Levy, E G Seidman, N Cerf-Bensussan, and M J Lentze Lipopolysaccharide modulation of normal enterocyte turnover by toll-like receptors is mediated by endogenously produced tumour necrosis factor {alpha} Gut, December 1, 2002; 51(6): 842 - 848. [Abstract] [Full Text] [PDF]
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M. Muroi and K.-i. Tanamoto The Polysaccharide Portion Plays an Indispensable Role in Salmonella Lipopolysaccharide-Induced Activation of NF-{kappa}B through Human Toll-Like Receptor 4 Infect. Immun., November 1, 2002; 70(11): 6043 - 6047. [Abstract] [Full Text] [PDF]
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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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A. C. Kirby, U. Yrlid, and M. J. Wick The Innate Immune Response Differs in Primary and Secondary Salmonella Infection J. Immunol., October 15, 2002; 169(8): 4450 - 4459. [Abstract] [Full Text] [PDF]
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M. A. Wolfert, T. F. Murray, G.-J. Boons, and J. N. Moore The Origin of the Synergistic Effect of Muramyl Dipeptide with Endotoxin and Peptidoglycan J. Biol. Chem., October 11, 2002; 277(42): 39179 - 39186. [Abstract] [Full Text] [PDF]
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D. Haller, M. P. Russo, R. B. Sartor, and C. Jobin IKKbeta and Phosphatidylinositol 3-Kinase/Akt Participate in Non-pathogenic Gram-negative Enteric Bacteria-induced RelA Phosphorylation and NF-kappa B Activation in Both Primary and Intestinal Epithelial Cell Lines J. Biol. Chem., October 4, 2002; 277(41): 38168 - 38178. [Abstract] [Full Text] [PDF]
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J. H. Wang, M. Doyle, B. J. Manning, Q. Di Wu, S. Blankson, and H. P. Redmond Induction of Bacterial Lipoprotein Tolerance Is Associated with Suppression of Toll-like Receptor 2 Expression J. Biol. Chem., September 20, 2002; 277(39): 36068 - 36075. [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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M. Muroi, T. Ohnishi, and K.-i. Tanamoto MD-2, a Novel Accessory Molecule, Is Involved in Species-Specific Actions of Salmonella Lipid A Infect. Immun., July 1, 2002; 70(7): 3546 - 3550. [Abstract] [Full Text] [PDF]
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T. Into, M. Fujita, T. Okusawa, A. Hasebe, M. Morita, and K.-I. Shibata Synergic Effects of Mycoplasmal Lipopeptides and Extracellular ATP on Activation of Macrophages Infect. Immun., July 1, 2002; 70(7): 3586 - 3591. [Abstract] [Full Text] [PDF]
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D. Iwaki, H. Mitsuzawa, S. Murakami, H. Sano, M. Konishi, T. Akino, and Y. Kuroki The Extracellular Toll-like Receptor 2 Domain Directly Binds Peptidoglycan Derived from Staphylococcus aureus J. Biol. Chem., June 28, 2002; 277(27): 24315 - 24320. [Abstract] [Full Text] [PDF]
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M. D. Lehner, F. Schwoebel, A. Kotlyarov, M. Leist, M. Gaestel, and T. Hartung Mitogen-Activated Protein Kinase-Activated Protein Kinase 2-Deficient Mice Show Increased Susceptibility to Listeria monocytogenes Infection J. Immunol., May 1, 2002; 168(9): 4667 - 4673. [Abstract] [Full Text] [PDF]
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H.-K. Lee, J. Lee, and P. S. Tobias Two Lipoproteins Extracted from Escherichia coli K-12 LCD25 Lipopolysaccharide Are the Major Components Responsible for Toll-Like Receptor 2-Mediated Signaling J. Immunol., April 15, 2002; 168(8): 4012 - 4017. [Abstract] [Full Text] [PDF]
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R. Berner, B. Furll, F. Stelter, J. Drose, H.-P. Muller, and C. Schutt Elevated Levels of Lipopolysaccharide-Binding Protein and Soluble CD14 in Plasma in Neonatal Early-Onset Sepsis Clin. Vaccine Immunol., March 1, 2002; 9(2): 440 - 445. [Abstract] [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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R. M. Wooten, Y. Ma, R. A. Yoder, J. P. Brown, J. H. Weis, J. F. Zachary, C. J. Kirschning, and J. J. Weis Toll-Like Receptor 2 Is Required for Innate, But Not Acquired, Host Defense to Borrelia burgdorferi J. Immunol., January 1, 2002; 168(1): 348 - 355. [Abstract] [Full Text] [PDF]
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E. Cario, D. Brown, M. McKee, K. Lynch-Devaney, G. Gerken, and D. K. Podolsky Commensal-Associated Molecular Patterns Induce Selective Toll-Like Receptor-Trafficking from Apical Membrane to Cytoplasmic Compartments in Polarized Intestinal Epithelium Am. J. Pathol., January 1, 2002; 160(1): 165 - 173. [Abstract] [Full Text] [PDF]
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S. O. Kim, K. Ono, and J. Han Apoptosis by pan-caspase inhibitors in lipopolysaccharide-activated macrophages Am J Physiol Lung Cell Mol Physiol, November 1, 2001; 281(5): L1095 - L1105. [Abstract] [Full Text] [PDF]
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M. Martin, J. Katz, S. N. Vogel, and S. M. Michalek Differential Induction of Endotoxin Tolerance by Lipopolysaccharides Derived from Porphyromonas gingivalis and Escherichia coli J. Immunol., November 1, 2001; 167(9): 5278 - 5285. [Abstract] [Full Text] [PDF]
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A. O. Aliprantis, D. S. Weiss, J. D. Radolf, and A. Zychlinsky Release of Toll-Like Receptor-2-Activating Bacterial Lipoproteins in Shigella flexneri Culture Supernatants Infect. Immun., October 1, 2001; 69(10): 6248 - 6255. [Abstract] [Full Text] [PDF]
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G. M. Scholz, S. D. Hartson, K. Cartledge, L. Volk, R. L. Matts, and A. R. Dunn The Molecular Chaperone Hsp90 Is Required for Signal Transduction by Wild-Type Hck and Maintenance of Its Constitutively Active Counterpart Cell Growth Differ., August 1, 2001; 12(8): 409 - 417. [Abstract] [Full Text] [PDF]
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H.-H. Mu, A. D. Sawitzke, and B. C. Cole Presence of Lpsd Mutation Influences Cytokine Regulation In Vivo by the Mycoplasma arthritidis Mitogen Superantigen and Lethal Toxicity in Mice Infected with M. arthritidis Infect. Immun., June 1, 2001; 69(6): 3837 - 3844. [Abstract] [Full Text] [PDF]
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S. Vogel, M. J. Hirschfeld, and P.-Y. Perera Signal integration in lipopolysaccharide (LPS)-stimulated murine macrophages Innate Immunity, June 1, 2001; 7(3): 237 - 241. [Abstract] [PDF]
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P. Muenzner, M. Naumann, T. F. Meyer, and S. D. Gray-Owen Pathogenic Neisseria Trigger Expression of Their Carcinoembryonic Antigen-related Cellular Adhesion Molecule 1 (CEACAM1; Previously CD66a) Receptor on Primary Endothelial Cells by Activating the Immediate Early Response Transcription Factor, Nuclear Factor-kappa B J. Biol. Chem., June 22, 2001; 276(26): 24331 - 24340. [Abstract] [Full Text] [PDF]
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J. da Silva Correia, K. Soldau, U. Christen, P. S. Tobias, and R. J. Ulevitch Lipopolysaccharide Is in Close Proximity to Each of the Proteins in Its Membrane Receptor Complex. TRANSFER FROM CD14 TO TLR4 AND MD-2 J. Biol. Chem., June 8, 2001; 276(24): 21129 - 21135. [Abstract] [Full Text] [PDF]
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S. Sanlioglu, C. M. Williams, L. Samavati, N. S. Butler, G. Wang, P. B. McCray Jr., T. C. Ritchie, G. W. Hunninghake, E. Zandi, and J. F. Engelhardt Lipopolysaccharide Induces Rac1-dependent Reactive Oxygen Species Formation and Coordinates Tumor Necrosis Factor-alpha Secretion through IKK Regulation of NF-kappa B J. Biol. Chem., August 3, 2001; 276(32): 30188 - 30198. [Abstract] [Full Text] [PDF]
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S. Viriyakosol, P. S. Tobias, R. L. Kitchens, and T. N. Kirkland MD-2 Binds to Bacterial Lipopolysaccharide J. Biol. Chem., October 5, 2001; 276(41): 38044 - 38051. [Abstract] [Full Text] [PDF]
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, The TLR4 cell line. Each
point represents the average of duplicate values. The experiment was
performed twice with similar results.
