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*
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;
Maxwell Finland Laboratory for Infectious Diseases, Boston Medical Center, Boston, MA 02118;
Norwegian University of Science and Technology, Trondheim, Norway; and
Pulmonary Center, Department of Medicine, Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B-dependent
reporter construct, endothelial leukocyte adhesion molecule CD25 (the
3E10 clone), were engineered to overexpress either human TLR2 or TLR4.
Transfected TLRs exhibited proper signaling functions, as evidenced by
increased LPS responsiveness of 3E10/TLR4 cells and acquisition of
sensitivity to TLR2-specific ligands upon transfection of TLR2 into
TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF
did not modulate TLR2 or TLR4 cell surface expression. Following LPS
exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable
decreases in LPS-mediated NF-B activation and mitogen-activated
protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment
profoundly inhibited LPS-induced NF-B translocation in Chinese
hamster ovary cells that concomitantly overexpressed human TLR4 and
myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4
or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with
LAM or STF decreased their NF-B responses induced by subsequent
stimulation with these substances or LPS. Conversely, prior exposure of
3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF,
indicating that LPS and mycobacterial products induce cross-tolerance.
Thus, tolerance to LPS and mycobacterial components cannot be
attributed solely to a decrease in TLR/MD-2 expression levels,
suggesting inhibition of expression or function of other signaling
intermediates. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
(5). Recognition of LPS and several other bacterial
products, including peptidoglycan (PGN), lipoteichoic acid (LTA), and
LAM is mediated by CD14 (6, 7, 8). However, CD14 lacks
transmembrane and intracellular domains necessary for signal
transduction and is now generally believed to bind various PAMPs and
present them to signal-transducing molecules that have been identified
as belonging to the Toll-like receptor (TLR) family. Indeed, using
fluorescence resonance energy transfer techniques, LPS was recently
shown to trigger a physical association between CD14 and TLR4
(9).
TLR family members are transmembrane proteins consisting of an
extracellular region that contains leucine-rich repeats flanked by
cysteine-rich clusters, a transmembrane portion, and a cytoplasmic
domain that is homologous to the signaling domain of the IL-1R type I
(10). Toll was initially shown to control dorsoventral
patterning during embryogenesis of Drosophila melanogaster
(11) and to mediate antifungal immune response in the
adult fly (12). Transfection of constitutively active Toll
leads to NF-B activation, cytokine production, and expression of
costimulatory molecules involved in the activation of adaptive immune
response through a pathway homologous to the NF-B signaling cascade
initiated by ligand engagement of the IL-1R type I (13).
Ten mammalian TLRs have been cloned to date, of which only three (TLR2,
TLR4, and TLR9) have known ligands (10, 13, 14, 15, 16, 17). A point
mutation within the tlr4 gene in the C3H/HeJ mouse strain
results in profound hyporesponsiveness to LPS (18, 19).
Another LPS-refractory mouse strain, C57BL10/ScCr, was reported to have
a deletion of the tlr4 gene (19, 20). These
studies, combined with results demonstrating acquisition of LPS
sensitivity upon TLR4 transfection of otherwise LPS-unresponsive cell
lines (21, 22, 23), and data on LPS hyporesponsiveness of TLR4
knockout mice (24), strongly support the hypothesis that
TLR4 is the primary signal-transducing receptor for enterobacterial
LPS. LPS-mediated signaling through TLR4 requires concomitant
expression of an accessory protein, myeloid differentiation protein-2
(MD-2), which physically associates with TLR4 (23, 25, 26, 27). TLR2 has also been reported to confer LPS
responsiveness upon its transfection into LPS-unresponsive HEK 293
cells (28, 29). However, this effect was likely to be due
to the presence of contaminating TLR2 ligands in commercial LPS
preparations as repurification of LPS completely abolished its ability
to activate TLR2-transfected cells, leaving TLR4-mediated responses
intact (22). In addition, LPS evokes biological responses
in Chinese hamster ovary (CHO)/CD14 cells that express endogenous
Chinese hamster TLR4 but not TLR2 (30). In contrast,
Dziarski et al. (31) have recently reported that
overexpression of TLR2, MD-2, and CD14 conferred upon HEK 293 cells
responsiveness to purified LPS or lipid A from Gram-negative bacteria
and showed a physical association of MD-2 with either TLR2 or TLR4.
Furthermore, recent data from our laboratory (32) and that
of Ulevitch and colleagues (33) have also demonstrated
that, in contrast to enterobacterial LPS, LPS purified from
Porphyromonas gingivalis and Leptospira
interrogans, respectively, signal via a TLR2-dependent mechanism.
Together, these results indicate that some endotoxins can activate TLR
proteins other than TLR4, although TLR4 appears to be the preeminent
receptor for enterobacterial LPS in primary cells. Live Mtb have been
demonstrated to signal via both TLR2 and TLR4, whereas their structural
components, LAM and STF, activate cells through TLR2 only (3, 4). Using transfection approaches and knockout techniques, TLR2
was shown to function as a pattern recognition receptor for diverse
bacteria and their products, including Gram-positive bacteria, PGN,
spirochetal pathogens Borrelia burgdorferi and
Treponema pallidum, as well as mycoplasma and their
lipopeptides (3, 4, 34, 35, 36, 37, 38, 39). Signaling initiated by TLR2
and TLR4 involves several common intracellular signaling intermediates,
including MyD88, IL-1R-associated kinases (IRAKs), and TNFR-associated
factor (TRAF)-6 that are also engaged in IL-1R type I-initiated NF-B
activation (40). However, LPS is capable of inducing
delayed NF-B and mitogen-activated protein (MAP) kinase activation
in macrophages derived from MyD88 knockout mice, indicating that
additional MyD88-independent signal transduction pathways also exist
(41).
Prior exposure to LPS significantly inhibits the ability of various
cell types to respond to subsequent LPS challenge. This phenomenon,
known as endotoxin tolerance, may protect the host from developing a
shock syndrome caused by excessive production of inflammatory cytokines
by host monocytes and macrophages hyperactivated by persisting
bacteria. LPS tolerance results in decreased activation of MAP
(42, 43, 44) and IB kinases (45), diminished
expression and activation of IRAK-1 (46, 47), inhibited
induction of transcription factors NF-B and AP-1 (44, 48), and suppressed gene expression and production of many
cytokines, including TNF-
, IL-1
, IL-6, and IL-12 (reviewed in
Ref. 49). However, expression of other mediators (e.g.,
IL-10, IL-1R antagonist, TNFRII, and NO) in LPS-tolerant cells is
either unaffected or may even be enhanced (49), suggesting
that endotoxin tolerance does not inhibit cellular functions globally,
but rather, represents a "reprogramming" of cells, possibly as a
means of adaptation to microbial infection (50). Although
molecular mechanisms of tolerance to microbial products have been
studied extensively, they still remain largely unknown. Inhibition of
the LPS signaling cascade involves upstream signaling intermediates,
e.g., decreased membrane GTP binding capacity and G protein content
(51), and altered expression of phospholipase C-
1 and
phosphatidylinositol-3'-kinase (52). Whereas endotoxin
tolerance does not affect cell surface expression of CD14
(53), LPS hyporesponsiveness induced by pretreatment of
mouse peritoneal macrophages with LPS was recently reported to decrease
cell surface expression of the TLR4-MD-2 receptor complex
(54). In contrast, other results suggest that modulation
of the downstream signal-transducing molecules of the TLR4 signaling
pathway is responsible for the development of an LPS-tolerant state
(44, 46, 47).
In the present work, we report that tolerance to LPS and mycobacterial
products can be induced in CHO/CD14 cells stably transfected with human
TLR2 or TLR4 under conditions where cell surface expression of these
TLRs is not influenced by exposure to microbial stimuli. Similarly,
prior exposure to endotoxin was found to inhibit LPS-mediated NF-B
translocation significantly in CHO cells that concomitantly
overexpressed TLR4 and MD-2, under conditions where cell surface
expression of both TLR4 and MD-2 was not down-regulated. Furthermore,
TLR4- (LPS) and TLR2- (LAM and STF) specific bacterial ligands were
found to induce a state of cross-tolerance analogous to that induced by
LPS and IL-1 in mouse macrophages (44). These data
indicate that down-regulation of TLR4/MD-2 cell surface
expression alone cannot account for the induction of tolerance to
microbial pathogens, and suggest involvement of additional ancillary
and/or downstream signaling intermediates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CHO fibroblast cell line stably transfected with a
NF-B-dependent reporter construct endothelial leukocyte adhesion
molecule CD25, the hygromycin vector pCEP4 (EL-1 cells), and the
EL-1/CD14 cell line (clone 3E10) were described previously (30, 34, 38, 55). Upon stimulation with LPS, TNF-, or IL-1, a
NF-B-dependent reporter plasmid drives the expression of surface
CD25 as a result of NF-B translocation. The 3E10/TLR2 cell line that
expresses Flag-tagged TLR2 was engineered by stable transfection of the
3E10 reporter cell line with the cDNA for human TLR2 in the pFLAG-CMV-1
vector as detailed previously (34, 38). The EL-1/TLR4 and
3E10/TLR4 cell lines were obtained by stable transfection of cells with
native human TLR4 cDNA inserted into the pcDNA3 expression vector
(provided by Dr. R. Medzhitov, Yale University, New Haven, CT) by the
calcium phosphate precipitation method (34, 38).
Transfectants were selected in G418, and a clonal cell line was
established subsequently by combining FACS cell sorting with the
anti-TLR4 mAb HTA125 (a gift from Dr. K. Miyake, Saga Medical
School, Saga, Japan) with limiting dilution cloning as described
previously (34, 38). The EL-1/TLR4/MD-2 cell line, which
concomitantly overexpresses native human TLR4 and Flag-tagged human
MD-2, was generated by stable transfection of EL-1/TLR4 cells with
Flag-tagged MD-2 cDNA cloned into the pEFBOS expression vector using
XhoI/BamHI sites (provided by Dr. K. Miyake).
Following cell sorting with M2 anti-Flag Ab, cell clones were
obtained by a standard limiting dilution cloning technique and
maintained in the presence of G418. 3E10 and EL-1 cells were cultured
in Ham’s F12 medium (BioWhittaker, Walkersville, MD) supplemented with
10% FBS (HyClone, Logan, UT), 2 mM L-glutamine,
100 U/ml penicillin, 100 µg/ml streptomycin, and 400 U/ml hygromycin
B (Calbiochem, San Diego, CA). EL-1/TLR4, EL-1/TLR4/MD-2, 3E10/TLR4,
and 3E10/TLR2 cell clones were maintained in the same medium
supplemented with 1 mg/ml G418 (Life Technologies, Gaithersburg, MD).
Table I
summarizes TLR expression
patterns of the cell lines used in this study. Protein-free,
phenol/water-extracted Escherichia coli LPS K235 was
prepared according to the method of McIntire et al. (56).
Arabinose-capped mycobacterial LAM was provided by Dr. J. Belisle
(Colorado State University, Fort Collins, CO) under the provisions of
National Institutes of Health Contract NO1 AI25147. Soluble
H37Ra-conditioned culture supernatant, termed STF, was prepared as
described (3). Levels of contaminating LPS in LAM and STF
preparations were determined using a Limulus lysate assay
(BioWhittaker) and were <1 pg/ml final concentration in all
experiments. Anti-Flag M2 mAb, mouse IgG1, mouse IgG2a, and
anti-mouse IgG (Fab-specific) FITC conjugate were purchased from
Sigma (St. Louis, MO), recombinant murine TNF- was from R&D Systems
(Minneapolis, MN), and anti-CD25 (anti-IL-2R) FITC conjugate
was obtained from BD Biosciences (San Jose, CA). Anti-ACTIVE MAP kinase
and c-Jun NH2-terminal kinase (JNK)
polyclonal Abs directed to phosphorylated forms of extracellular
signal-regulated kinase (ERK) 1/2 and JNK 1/2, and
donkey-anti-rabbit IgG (H + L) HRP conjugate were obtained from
Promega (Madison, WI); phospho-p38 MAP kinase Ab and p38 MAP
kinase Ab were purchased from New England Biolabs (Beverly, MA). Rabbit
polyclonal IgG against IB- (C21) was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA).
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Cells were plated in 12-well tissue culture plates at a density of 3 x 105 cells/well in respective selective medium and incubated overnight at 37°C in a 5% CO2 atmosphere. Thereafter, cells were washed three times with PBS, resuspended in medium, and pretreated for 5 h with various bacterial components. Following three washes with PBS, cells were restimulated for 18 h with medium, LPS, or mycobacterial components, detached from the plastic with trypsin/EDTA, and examined by FACS analysis for the presence of surface CD25 as described (3, 4, 34, 38, 55). Flag-tagged proteins were detected by FACS analysis with anti-Flag M2 mAb as described (3, 4, 34, 38). Native human TLR4 expression was measured by staining cells for 45 min with anti-TLR4 mAb HTA125 or mouse IgG2a isotype control Ab (5 µg/ml each) on ice, washing twice with PBS/2% FCS, and incubating for 30 min with secondary FITC-labeled anti-mouse IgG (5 µg/ml). Thereafter, cells were subjected to flow cytometry analysis on an Epics-Coulter cell analyzer (Coulter, Miami, FL).
Preparation of nuclear extracts and EMSA
Cells were plated into six-well plates (Costar) at a density of
2 x 106 cells/well, grown overnight, washed
with PBS, and treated as described in the figure legends. Nuclear
extracts were prepared as described previously (44).
Protein concentration was determined using the Bio-Rad assay kit
(Bio-Rad, Hercules, CA). The NF-B-specific oligonucleotide probe
5'-AGTTGAGGGGACTTTCCCAGGC-3' from the murine Ig-B light chain gene
enhancer was synthesized by the BIC Synthesis and Sequencing Facility
(Uniformed Services University of the Health Sciences, Bethesda, MD)
and 32P-end-labeled with T4 polynucleotide kinase
(Promega) as recommended by the manufacturer. Nuclear extracts (4 µg)
were incubated with 0.2 ng radiolabeled DNA probe in a binding buffer
(final volume 20 µl) containing 2 µg poly(dI-dC) (Amersham
Pharmacia Biotech, Piscataway, NJ), 10 mM Tris-HCl (pH 7.9), 50 mM KCl,
4% glycerol, 1 mM DTT, and 0.25 mg/ml BSA for 30 min at room
temperature. After incubation, a portion of each reaction (18 µl) was
loaded onto a 5% nondenaturing polyacrylamide gel, and the DNA-protein
complexes were resolved from free oligonucleotide by electrophoresis
(0.25 x Tris borate/EDTA, 150 V, 2 h). The gels were dried
(80°C, 2 h) and exposed to x-ray films (X-OMAT AR; Eastman
Kodak, Rochester, NY).
Western blot analysis
Cellular extracts were prepared as described (57). Twenty micrograms of total protein was added in Laemmli buffer, boiled for 5 min, resolved by SDS-12% PAGE in Tris/glycine/SDS buffer (25 mM Tris, 250 mM glycine, 0.1% SDS), and transferred onto Immobilon P transfer membranes (Millipore, Bedford, MA) (100 V, 1.5 h, 4°C). After blocking for 2 h in TBS-T (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat milk, membranes were washed three times in TBS-T and probed for 20 h at 4°C with the respective Abs (dilution 1/1000 in TBS-T/0.5% nonfat milk). Following washing in TBS-T, membranes were incubated with secondary HRP-conjugated, donkey anti-rabbit IgG (1/10,000 dilution) and washed five times in TBS-T, and bands were detected using ECL reagents (Amersham Pharmacia Biotech) according to manufacturer’s directions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Induction of LPS tolerance has been reported to be accompanied by
diminished staining of mouse macrophages with a mAb that detects an
epitope defined by the interaction of TLR4 with MD-2 (54).
Hence, we first sought to examine whether overexpression of TLR4 could
compensate for such an LPS-mediated decrease in TLR4-MD-2 expression,
resulting in a reduced ability of LPS to cause endotoxin tolerance. As
TLR2 has been reported to confer responsiveness to a large variety of
Gram-positive, fungal, and mycobacterial structures (3, 4, 34, 35, 36, 37, 38, 39), the effect of TLR2 overexpression on the induction of
tolerance to LPS and mycobacterial components was also studied. As a
model system, we used the CHO/CD14 fibroblast cell line that expresses
a reporter plasmid comprised of the human CD25 reporter gene under the
control of a NF-B-dependent endothelial leukocyte adhesion
molecule-1 promoter (the 3E10 cell clone). This reporter cell line was
further stably transfected with the plasmids containing either native
human TLR4 cDNA or Flag-tagged human TLR2 cDNA under control of a
strong, constitutively active, CMV promoter. Clonal 3E10/TLR4 and
3E10/TLR2 cell lines were obtained by combining cell sorting and
limiting dilution cloning techniques. FACS analysis with anti-Flag
M2 mAb or anti-TLR4 HTA125 mAb confirmed high levels of cell
surface expression of the transfected TLRs, whereas the control cell
line 3E10 showed no staining with either anti-Flag M2 (Fig. 1) or HTA125 mAb (data not shown). The
expression of transfected TLRs in 3E10/TLR4 and 3E10/TLR2 cells was
also confirmed by RT-PCR with primers specific for human TLR4 and TLR2
(data not shown). As measured by FACS analysis, treatment of 3E10/TLR4
or 3E10/TLR2 cells with LPS for 5 h did not alter cell surface
levels of transfected TLRs when compared with those detected after
treatment with medium alone (Fig. 1). Likewise, no change in the
expression of transfected TLRs was seen after restimulation with LPS
for 20 h or after pretreatment or restimulation with LAM or STF
(data not shown).
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MAP kinases play an important role in mediating many cell
functions, including activation of various transcription factors and
production of pro- and anti-inflammatory cytokines
(58). Therefore, we sought to examine whether
overexpression of TLR2 or TLR4 would affect the capacity of LPS to
induce an endotoxin-tolerant state as reflected by decreased MAP kinase
activation. Because phosphorylation of the MAP kinases closely
correlates with their activation (59), MAP kinase
phosphorylation was measured in cellular extracts by Western analysis
with Abs specific for phosphorylated forms of the respective MAP
kinases. Similar to our previous findings in mouse macrophages
(44), stimulation of medium-pretreated 3E10, 3E10/TLR2,
and 3E10/TLR4 cell lines with 10 ng/ml LPS resulted in phosphorylation
of the JNK 1/2 and p38 MAP kinases (Fig. 2). However, in contrast to macrophages,
no ERK 1/2 phosphorylation was seen after treatment of any of these
three cell lines with LPS (data not shown). These results are
consistent with our previously published observation that LPS fails to
induce tyrosine phosphorylation of proteins with molecular mass
corresponding to ERK 1/2 MAP kinases in CHO/CD14 cells, whereas PMA
induced a strong response (60). Overexpression of human
TLR4 in 3E10 cells led to enhanced phosphorylation of JNK 1/2 and p38
MAP kinases in response to stimulation with 10 ng/ml LPS compared with
the responses detected in the parental 3E10 cell line and in 3E10/TLR2
cells (Fig. 2). Pretreatment of 3E10, 3E10/TLR2, and 3E10/TLR4 cell
lines for 5 h with 10 ng/ml LPS led to a complete abrogation of
the ability of these cells to respond to subsequent LPS challenge with
phosphorylation of JNK 1/2 and p38 MAP kinases (Fig. 2). Comparable
levels of total p38 MAP kinase expression were detected in all three
cells lines that were not modulated by LPS (Fig. 2), indicating that
the observed changes in MAP kinase phosphorylation were not due to
unequal protein loading. Thus, overexpression of either TLR2 or TLR4 in
3E10 cells does not affect the capacity of LPS to induce an
endotoxin-tolerant state at the level of JNK and p38 activation.
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B activation in
3E10, 3E10/TLR2, and 3E10/TLR4 cell clones
The next experiments were conducted to examine whether
overexpression of TLR2 or TLR4 affects the induction of endotoxin
tolerance, as measured by suppressed NF-B activation in response to
LPS challenge. We first assessed the effect of LPS pretreatment on
LPS-mediated degradation of IB- in 3E10, 3E10/TLR2, and 3E10/TLR4
cell clones. Stimulation of medium-pretreated 3E10, 3E10/TLR2, and
3E10/TLR4 cells for 30 min with 1 ng/ml LPS resulted in a partial
degradation of IB-, whereas a complete degradation was reached
when LPS was used at a concentration of 10 ng/ml (Fig. 3). In contrast, LPS pretreatment of all
three cell lines almost completely abolished LPS-induced IB-
degradation (Fig. 3). To confirm and extend these results, we examined
LPS-induced NF-B activation in medium- or LPS-pretreated 3E10,
3E10/TLR2, and 3E10/TLR4 cells based on NF-B DNA binding activity
and on the ability to transactivate NF-B-dependent expression of the
CD25 reporter gene. As depicted in Fig. 4, EMSA analyses of 3E10 and 3E10/TLR2
cells showed a suboptimal NF-B response following their stimulation
with 0.1 ng/ml LPS, which reached a plateau at 10 and 100 ng/ml LPS.
Overexpression of TLR4 enhanced LPS-mediated NF-B translocation
10-fold, because as little as 1 ng/ml LPS was required to induce
maximal levels of NF-B translocation in the 3E10/TLR4 cell line
(Fig. 4). Pretreatment with LPS led to a >100-fold shift of the LPS
dose-response curve to the right in all three cell lines (Fig. 4),
indicating a significant decrease in LPS-induced NF-B translocation.
In 3E10, 3E10/TLR4 (data not shown), and 3E10/TLR2 (Fig. 8) cells, TNF--mediated NF-B
translocation was not influenced by prior exposure to LPS, and,
conversely, pretreatment of these cell lines with TNF- did not
affect LPS-induced NF-B translocation (data not shown). To measure
the transactivating potential of NF-B, CD25 cell surface expression
was subsequently measured in either the parental 3E10 cell line or in
3E10/TLR2 and 3E10/TLR4 clones. LPS stimulation of medium-pretreated
3E10, 3E10/TLR2, and 3E10/TLR4 cell lines resulted in a potent
induction of CD25 cell surface expression (Fig. 5, A–C, top).
In all three cell lines, prior exposure
to LPS led to significantly diminished expression of membrane CD25 in
response to LPS restimulation (Fig. 5, A–C,
bottom), consistent with suppressed LPS-mediated IB-
degradation and NF-B translocation observed in LPS-tolerant cells.
Notably, TNF--induced NF-B-dependent CD25 cell surface expression
was similar in these cell lines and was not affected by LPS
pretreatment (data not shown), indicating specific down-regulation of
the LPS signaling pathways in LPS-tolerant cells. Consistent with
suppressed NF-B activation observed in 3E10, 3E10/TLR2, and
3E10/TLR4 cells, LPS pretreatment also led to a marked inhibition of
LPS-inducible endogenous IL-6 gene expression in all three cell lines
(data not shown). Thus, overexpression of TLR2 or TLR4 in 3E10 cells
does not affect the capacity of LPS to induce a state of endotoxin
tolerance.
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Thus far, our data suggest that down-regulation of TLR4 expression
is not the mechanism underlying early endotoxin tolerance. Because the
previously published results implying this mechanism were obtained with
an Ab that detected a TLR4/MD-2-shared epitope (54), we
next sought to determine whether overexpression of MD-2 would diminish
the ability of TLR4-expressing cells to become tolerant to LPS. To this
end, CD14-negative CHO cells were stably transfected with native human
TLR4 and Flag-tagged human MD-2 and their LPS responses were compared
with those seen in the parental EL-1 cell line or in EL-1 cells
engineered to overexpress human TLR4 only. Cell surface expression of
TLR4 was clearly detectable in both EL-1/TLR4 and EL-1/TLR4/MD2 cells
by FACS analysis with anti-TLR4 mAb HTA125, whereas staining with
anti-Flag Ab M2 verified that MD-2 was expressed only on the cell
surface of the EL-1/TLR4/MD-2 cell line (Fig. 6, A and B, top).
Interestingly, concomitant expression of TLR4 and MD-2 significantly
enhanced the fluorescence intensity of TLR4 compared with that measured
in EL-1/TLR4 cells. Functional analyses demonstrated that in the
absence of CD14, LPS failed to induce NF-B translocation in the
parental EL-1 cell line even when used at a concentration as high as 1
µg/ml (data not shown). Overexpression of TLR4 in CD14-negative CHO
cells enabled NF-B activation in response to 1000 ng/ml LPS, whereas
cotransfection of TLR4 and MD-2 conferred upon CHO cells the ability to
respond to as little as 1 ng/ml LPS (Fig. 6, A and B,
bottom). Fig. 6, A and B, shows that
pretreatment of both EL-1/TLR4 and EL-1/TLR4/MD2 cells with 10 ng/ml
LPS for 5 h did not significantly modulate expression of TLR4 or
MD-2, whereas LPS-mediated NF-B activation was dramatically
inhibited in both cell lines, indicating the induction of endotoxin
tolerance. In contrast, stimulation with TNF- resulted in comparable
NF-B translocation in both cell lines regardless of whether they
were pretreated with LPS or medium (Fig. 6, A and B,
bottom), demonstrating specific inhibition of the LPS signaling
pathway in endotoxin-tolerant cells. These data indicate that
concomitant expression of TLR4 and MD-2 in CHO cells failed to prevent
the induction of endotoxin tolerance.
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Next, we examined whether the mycobacterial TLR2 ligands, LAM and
STF, can elicit tolerance against themselves in 3E10 cells that
overexpress human TLR2. In addition, the abilities of TLR4- (LPS) and
TLR2- (LAM, STF) specific bacterial products to induce cross-tolerance
were studied. Neither LAM (Fig. 7A) nor STF (data not shown)
induced NF-B-dependent CD25 expression in the 3E10/TLR4 cell line
that expresses functional TLR4, but not TLR2 (30),
whereas strong NF-B activation was seen in response to LPS.
Similarly, no NF-B translocation was elicited by LAM or STF in the
parental 3E10 cell line, which lacks functional endogenous TLR2,
whereas marked NF-B activation was induced by both LPS and TNF-
(Fig. 8
, A and B).
Confirming previously published observations (3, 4, 34),
overexpression of TLR2 rendered 3E10 cells sensitive to both LAM and
STF, as evidenced by NF-B-dependent CD25 expression (Fig. 7B and data not shown) and NF-B translocation (Fig. 8, A and B). Pretreatment of 3E10/TLR2 cells with
LPS, LAM, or STF for 5 h reduced their capacity to exhibit NF-B
responses upon restimulation with any of these bacterial structures
(Figs. 7B and 8, A and B). Of note,
the induction of cross-tolerance was observed despite the failure of
these bacterial products to affect cell surface expression of TLR2
(Fig. 1 and data not shown). In contrast, prior exposure of the
3E10/TLR4 cell line to LAM or STF did not change LPS-mediated CD25 cell
surface expression (Fig. 7A and data not shown) or NF-B
translocation (Fig. 8, A and B), whereas LPS
pretreatment markedly decreased NF-B activation induced by
subsequent LPS challenge. LPS pretreatment of 3E10/TLR2 cells
significantly inhibited NF-B activation in response to subsequent
stimulation with LPS, LAM, or STF, whereas pretreatment with LAM or STF
suppressed LPS-mediated NF-B activation to a lesser extent than that
induced by restimulation with LAM or STF (Figs. 7B and 8,
A and B). Thus, pretreatment of 3E10/TLR2 cells
with either a TLR4-specific bacterial ligand (LPS) or TLR2-specific
mycobacterial components, LAM and STF, results in the development of
cross-tolerance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation was also observed following a much
shorter exposure of mouse macrophages to LPS (1 h), conditions, where
cell surface expression of the TLR4-MD-2 receptor complexes was
diminished only partially (54). Furthermore, because the
mAb used in this study was directed against the complex of TLR4 with
MD-2, it is unclear whether expression of both TLR4 and MD-2 is
decreased, only TLR4 or MD-2 is down-regulated, TLR4-MD-2 association
is disrupted, or whether a protein modification induced by LPS
pretreatment masks the immunoreactive epitope. In the current study, we reasoned that if tolerance to microbial pathogens occurs mainly due to down-regulation of their signaling receptors, then overexpression of TLR2 or TLR4 should reduce the ability of TLR2- and TLR4-specific bacterial products to induce tolerance. To address this question, clonal 3E10/TLR2 and 3E10/TLR4 cell lines were obtained following stable transfection of the parental 3E10 cell clone with expression plasmids that contain human TLR2 or TLR4 cDNA under the control of a strong constitutively active CMV promoter. In line with previously published observations (3, 4), transfection of TLR2 into 3E10 cells, which express functional endogenous TLR4 but not TLR2 (30), imparted responsiveness to TLR2-specific mycobacterial components LAM and STF. Proper functional activities of transfected TLR4 were evidenced by increased LPS sensitivity of 3E10/TLR4 transfectants, confirming earlier reports on augmentation of cell responsiveness to LPS upon an increase in expression levels of cell surface TLR4 (61, 62). Importantly, FACS analyses showed that LPS, LAM, or STF did not influence cell surface expression of transfected TLR2 or TLR4. Thus, even if LPS might transiently decrease expression of endogenous TLR4, the preponderance of heterologously expressed human TLR4 molecules, whose expression is not affected by LPS, would ensure proportionately lower down-regulation of total TLR4 in 3E10/TLR4 cells compared with the 3E10 cell line. In turn, this should result in a marked difference in the capacities of 3E10/TLR4 and 3E10 cell lines to become tolerant to LPS. In contrast, the absence of functional, endogenous TLR2 in 3E10 cells creates a situation where induction of tolerance to TLR2-specific mycobacterial products would occur despite their failure to affect cell surface expression of transfected human TLR2.
A number of observations presented herein suggest
that tolerance to LPS and mycobacterial products can be induced
independently of down-regulation of cell surface expression of TLR2 and
TLR4. First, both the parental 3E10 cell line and 3E10 cell clones that
overexpress TLR2 or TLR4 showed comparable inhibition of various
LPS-induced responses following LPS pretreatment, including MAP kinase
phosphorylation, NF-B activation, and IL-6 gene expression. Second,
not only LPS, but also TLR2-specific LAM and STF, rendered 3E10/TLR2
cells tolerant to these microbial products. These data extend a
previously reported observation on the induction of LAM tolerance in
human monocytes by LAM (63) and demonstrate that in
3E10/TLR2 cells, tolerance to LAM or STF can be induced despite the
failure of mycobacterial products to affect TLR2 expression. Third,
LPS, LAM, and STF were found to induce cross-tolerance in the 3E10/TLR2
cell line. Similar findings have been reported in other models of
cross-tolerance using distinct TLR2 bacterial ligands, including
muramyl dipeptide and mycoplasma-derived macrophage-activating
lipopeptide, 2 kDa (MALP-2) (64, 65). Furthermore, Lehner
et al. (66) have recently demonstrated the induction of
cross-tolerance in mouse macrophages by LPS and highly purified LTA
that have been shown in this report to act via TLR4 and TLR2,
respectively. Our results extend these observations and indicate that
engagement of the TLR2 signaling pathway by prior exposure to
mycobacterial products that "bypass" the TLR4 receptor stage
inhibits TLR4-mediated signal transduction events initiated by LPS.
Fourth, this study shows that prior exposure of 3E10/TLR2 cells to LPS
down-regulated NF-B activation induced not only by LPS, but also by
the TLR2 ligands LAM and STF. Of note, LPS pretreatment caused
comparable decreases in NF-B activation in response to LPS, STF, or
LAM, whereas the TLR2-specific mycobacterial products potently induced
tolerance against themselves and only partially inhibited LPS-mediated
NF-B translocation. Similar to our data, prior exposure of mouse
macrophages to LPS has been shown to suppress significantly both LPS-
and LTA-induced TNF- release, whereas LTA pretreatment strongly
inhibited TNF- production in response to LTA but was a weaker
inducer of LPS tolerance (66). Sato et al.
(65) also reported that LPS pretreatment rendered mouse
macrophages tolerant to LPS and to MALP-2, although in their system,
LPS-mediated induction of tolerance to MALP-2 was less effective
compared with significant suppression of LPS responses observed in
endotoxin-tolerant cells. The reason for these quantitative differences
in the induction of cross-tolerance among LPS and various TLR2 ligands
(LAM, STF, LTA, and MALP-2) is unclear but might be related to
their differential use of other TLRs that could associate with TLR2 or
TLR4. Indeed, a dominant-negative mutant of TLR6 was reported to
inhibit TNF- production in response to TLR2-specific PGN, but not to
another TLR2 ligand, the synthetic tripalmitoylated lipopeptide
Pam3CSK4
(67).
MD-2 is required for enabling LPS-mediated signal transduction via TLR4
(23, 25, 26, 27). Hence, LPS tolerance may occur due to
down-regulation of endogenous MD-2, leading to suppression of TLR4
signal transduction. However, if this were the case, then
overexpression of MD-2, together with TLR4, would be predicted to
inhibit the induction of LPS tolerance. To examine this possibility,
CD14-negative CHO cells (the EL-1 cell clone) were stably transfected
with either native human TLR4 alone or concomitantly with TLR4 and
MD-2, and the effect of LPS pretreatment on the expression of TLR4 and
MD-2, as well as on functional responses of transfected cell lines, was
evaluated. FACS analyses of the EL-1/TLR4/MD-2 cell line revealed
significantly higher fluorescence intensity of TLR4 compared with that
detected in the EL-1/TLR4 cells, extending similar results obtained
from a comparative analysis of human HEK 293/TLR4/MD-2 vs 293/TLR4
cells (31, 68). The mechanism by which MD-2 expression
facilitates the detection of TLR4 is unknown. It is interesting that a
MD-2 related protein, MD-1, binds to RP105, a member of the TLR family
expressed on B lymphocytes (69), and is thought to
regulate RP105 cell surface expression (70). Analogous to
MD-1, MD-2 may bind to TLR4, stabilizing its expression on the cell
surface, which could facilitate TLR4 detection and signal transduction.
To study consequences of overexpression of TLR4 and MD-2 on cell
responsiveness to LPS, we undertook EMSA analysis of NF-B
translocation. In the absence of CD14, LPS failed to induce NF-B
translocation in EL-1 cells despite the presence of endogenous TLR4
(data not shown), consistent with an important role for membrane
CD14 in mediating LPS responsiveness (7). Even though
cells were stimulated in the presence of FCS that contains
soluble CD14, soluble CD14 does not confer LPS sensitivity upon
CHO cells (60). In agreement with previously published
data in HEK 293 cells (21), overexpression of TLR4 alone
in CHO cells induced NF-B translocation only in response to high
concentrations of LPS, whereas cotransfection of TLR4 together with
MD-2 significantly enhanced LPS-mediated NF-B activation. Of
importance, LPS pretreatment markedly suppressed LPS-mediated NF-B
translocation in both EL-1/TLR4 and EL-1/TLR4/MD-2 cell lines despite
the failure of LPS to decrease cell surface expression of either TLR4
or MD-2. It could be argued that stable transfection of CHO cells
with TLR4 and MD-2 may produce a large number of these molecules that
are not down-regulated by LPS and are expressed on the cell surface
without being associated in a functional signal-transducing complex.
However, MD-2 is a secreted protein that has neither a transmembrane
domain nor GPI linkage capable of anchoring this protein to the cell
surface (25, 27). Therefore, MD-2 would be predicted to be
present on the cell surface only if associated with TLR4, assuming that
TLR4 is its only binding partner. Indeed, when MD-2 was expressed
alone, its cell surface expression was undetectable by FACS, whereas
cotransfection of MD-2 along with TLR4 resulted in MD-2 cell surface
expression (25). Furthermore, several reports have
demonstrated coimmunoprecipitation of MD-2 with TLR4 (27, 31, 68). Using coimmunoprecipitation, we have also been able to show
physical interaction of TLR4 with MD-2 in EL-1/TLR4/MD-2 cells (data
not shown). Altogether, our results indicate that endotoxin tolerance
can be induced in CHO cells that overexpress TLR2, TLR4, or TLR4 plus
MD-2, whose expression levels are not subject to LPS regulation. Thus,
it seems unlikely that down-regulation of TLR4/MD-2 complexes only
underlies the phenomenon of endotoxin tolerance. Indeed, we
demonstrated previously that LPS and IL-1 induce cross-tolerance in
mouse macrophages (44), yet IL-1 pretreatment does not
decrease TLR4-MD-2 cell surface expression (54). As IL-1
and LPS signal through different receptors but share many downstream
signaling intermediates (71), it suggests the existence of
additional mechanisms of endotoxin tolerance. Furthermore, if the
mechanism of LPS tolerance were due solely to a decrease in the number
of TLR4 and/or MD-2, then all LPS responses should be inhibited in
endotoxin-tolerant cells, as TLR4 has been unambiguously demonstrated
as the principal LPS receptor in mammals (18, 19, 20, 21, 22, 23, 24, 25, 26, 27). This is
not the case, because a number of cellular responses are not affected
or even enhanced in LPS-tolerant cells (reviewed in Ref.
49). In contrast, induction of LPS cross-tolerance by
mycobacterial products could result from a decrease in cell surface
expression of endogenous TLR4 and/or MD-2 following pretreatment of
3E10/TLR2 cells with LAM or STF, providing that cross-talk between the
TLR2 and TLR4 signaling pathways exists. However, Sato et al.
(65) found that prior exposure of mouse macrophages to
TLR2-specific MALP-2 failed to decrease the expression of TLR4-MD-2
complex. In addition, overexpression of MD-2 in RAW 264.7 macrophages
did not affect the ability of the TLR2-reactive mycobacterial component
LAM to elicit cellular responses, while greatly enhancing cell
sensitivity to LPS (72). Furthermore, a LPS-unresponsive
CHO cell clone that expresses a mutant MD-2 protein was found to
respond normally to Mtb (72). Therefore, although MD-2 may
promote optimal signal transduction by LPS and PGN (23, 25, 26, 27, 31), it does not appear to influence TLR2-mediated cellular
responses to mycobacterial components. Taken together, our results
further argue against the hypothesis that tolerance to mycobacterial
pathogens occurs due to down-regulation of TLR2 or MD-2 expression.
We propose that tolerance to bacterial products targets intracellular
signaling intermediates downstream of TLRs. Following activation of
TLR2 or TLR4, a common set of signaling intermediates participates in
mediating NF-B activation, including MyD88, IRAK-1, TRAF-6,
evolutionary conserved signaling intermediate in Toll pathways,
NF-B-inducing kinase, and IB kinases (24, 29, 39, 40, 41, 73, 74). Decreased activity of IRAK-1 has been reported in
LPS-tolerant THP-1 human monocytic cells (47) and
mouse Kupffer cells (46). Furthermore, IRAK-1 failed to
associate with MyD88 in THP-1 cells pretreated with LPS, whereas the
association of phosphorylated IRAK-1 with TRAF-6 was not affected
(47), suggesting IRAK-1 as an attractive target for
down-regulation as a consequence of LPS tolerance. Expression and
functions of other downstream signaling molecules, e.g., MyD88, TRAF-6,
and evolutionary conserved signaling intermediate in Toll pathways,
could also be inhibited in cells rendered tolerant to LPS or
TLR2-specific mycobacterial ligands. In contrast, some responses that
are not dependent on MyD88 or IRAK-1 (41) could be even
enhanced in tolerant cells, whereas MyD88- and IRAK-1-dependent
responses may be down-regulated. Studies are in progress to evaluate
this theory and to identify downstream signaling intermediates involved
in the mechanisms of tolerance to LPS and mycobacterial components.
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Acknowledgments |
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Footnotes |
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2 The opinions or assertions contained within are the private views of the authors and should not be construed as official or necessarily reflecting the views of the Uniformed Services University of the Health Sciences or the Department of Defense.
3 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814. E-mail address: vogel{at}bob.usuhs.mil
4 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; Mtb, Mycobacterium tuberculosis; LAM, lipoarabinomannan; STF, soluble tuberculosis factor; MD-2, myeloid differentiation protein-2; LTA, lipoteichoic acid; PGN, peptidoglycan; IRAK, IL-1R-associated kinase; TRAF, TNFR-associated factor; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MALP-2, macrophage-activating lipopeptide, 2 kDa; CHO, Chinese hamster ovary.
Received for publication January 5, 2001. Accepted for publication June 21, 2001.
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A. E. Medvedev, A. Lentschat, L. M. Wahl, D. T. Golenbock, and S. N. Vogel Dysregulation of LPS-Induced Toll-Like Receptor 4-MyD88 Complex Formation and IL-1 Receptor-Associated Kinase 1 Activation in Endotoxin-Tolerant Cells J. Immunol., November 1, 2002; 169(9): 5209 - 5216. [Abstract] [Full Text] [PDF]
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M. L. Vandenplas, R. W. Carlson, B. S. Jeyaretnam, B. McNeill, M. H. Barton, N. Norton, T. F. Murray, and J. N. Moore Rhizobium Sin-1 Lipopolysaccharide (LPS) Prevents Enteric LPS-induced Cytokine Production J. Biol. Chem., October 25, 2002; 277(44): 41811 - 41816. [Abstract] [Full Text] [PDF]
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M. Adib-Conquy and J.-M. Cavaillon Gamma Interferon and Granulocyte/Monocyte Colony-stimulating Factor Prevent Endotoxin Tolerance in Human Monocytes by Promoting Interleukin-1 Receptor-associated Kinase Expression and Its Association to MyD88 and Not by Modulating TLR4 Expression J. Biol. Chem., July 26, 2002; 277(31): 27927 - 27934. [Abstract] [Full Text] [PDF]
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S. Sato, O. Takeuchi, T. Fujita, H. Tomizawa, K. Takeda, and S. Akira A variety of microbial components induce tolerance to lipopolysaccharide by differentially affecting MyD88-dependent and -independent pathways Int. Immunol., July 1, 2002; 14(7): 783 - 791. [Abstract] [Full Text] [PDF]
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R. Jacinto, T. Hartung, C. McCall, and L. Li Lipopolysaccharide- and Lipoteichoic Acid-Induced Tolerance and Cross-Tolerance: Distinct Alterations in IL-1 Receptor-Associated Kinase J. Immunol., June 15, 2002; 168(12): 6136 - 6141. [Abstract] [Full Text] [PDF]
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A. S. Cross Invited review: Endotoxin tolerance -- current concepts in historical perspective Innate Immunity, April 1, 2002; 8(2): 83 - 98. [PDF]
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 M. W. Hornef, T. Frisan, A. Vandewalle, S. Normark, and A. Richter-Dahlfors Toll-like Receptor 4 Resides in the Golgi Apparatus and Colocalizes with Internalized Lipopolysaccharide in Intestinal Epithelial Cells J. Exp. Med., February 25, 2002; 195(5): 559 - 570. [Abstract] [Full Text] [PDF]
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J. da Silva Correia and R. J. Ulevitch MD-2 and TLR4 N-Linked Glycosylations Are Important for a Functional Lipopolysaccharide Receptor J. Biol. Chem., January 11, 2002; 277(3): 1845 - 1854. [Abstract] [Full Text] [PDF]
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