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*
Laboratory of Host Defense and Germfree Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, Japan; and
Department of Oral Microbiology, Asahi University School of Dentistry, Gifu, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-
,
or TNF-
significantly increased TLR2 mRNA expression, whereas TLR4
mRNA expression remained constant. TLR2 mRNA increase in response to
synthetic lipid A was severely impaired in splenic macrophages isolated
from TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential
role in the process. Specific inhibitors of mitogen-activated
protein/extracellular signal-regulated kinase kinase and p38 kinase did
not significantly inhibit TLR2 mRNA up-regulation by LPS. In contrast,
LPS-mediated TLR2 mRNA induction was abrogated by pretreatment with a
high concentration of curcumin, suggesting that NF-
B activation may
be essential for the process. Taken together, our results indicate that
TLR2, in contrast to TLR4, can be induced in macrophages in response to
bacterial infections and may accelerate the innate immunity against
pathogens. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B (2, 3), AP-1
(3), extracellular signal-regulated kinases
(ERKs)3 (4, 5), c-Jun N-terminal kinases (JNKs) (6, 7), and p38
kinases (8, 9). CD14, a cell surface protein, and LPS
binding protein, a serum factor, bind LPS with high affinity, mediating
LPS responses (10, 11, 12). As CD14 is a
glycosylphosphatidylinositol-anchored protein, the LPS receptor complex
was assumed to contain an additional signaling component. Toll, first identified as a protein controlling dorsoventral pattern formation in the early development of Drosophila (13), has been shown to participate in antimicrobial immune responses (14). Toll is conserved throughout various species encoding a transmembrane protein of which the intracellular domain is homologous to that of the IL-1 receptor family proteins (13). Recently, several mammalian Toll homologues have been identified and termed Toll-like receptors (TLRs) (15). In addition to their cytoplasmic tails, the newly identified receptors share repeating leucine-rich motifs in their extracellular regions. In the past few years two of the mammalian TLRs, TLR2 and TLR4, have been shown to mediate the LPS responsiveness in in vitro transfection systems (16, 17, 18, 19, 20). Although TLR2 is capable of mediating LPS signals in vitro, its role as an LPS receptor in vivo has been questioned as a result of the recent findings that two mouse strains (C3H/HeJ and C57BL10/ScCr) that exhibit impaired ability to respond to many types of LPS have different mutations in the TLR4 gene (21, 22). Also, gene-disrupted mice of TLR4, but not TLR2, demonstrate phenotypes similar to those of LPS-hyporesponsive strains (23). These findings suggest that TLR4 is the dominant receptor for at least some types of LPS, whereas TLR2 is dispensable. In contrast, TLR2 has recently been suggested to mediate signals from other bacterial components, including lipoteichoic acid, peptidoglycan, and lipoproteins/lipopeptides (24, 25, 26, 27).
In this study, we examined the gene expression of TLR2 and TLR4 in
mouse macrophages. An intraperitoneal injection of LPS significantly
increased the mRNA levels of TLR2, but not TLR4, in splenic
macrophages. Treatment of mouse macrophage cell lines with LPS,
synthetic lipid A, IL-2, IL-15, IL-1, IFN-, or TNF-
significantly increased TLR2 mRNA expression, whereas TLR4 mRNA levels
remained constant. The TLR2 mRNA increase in response to synthetic
lipid A was severely impaired in splenic macrophages isolated from
TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential role
in this process. Specific inhibitors of mitogen-activated protein
(MAP)/ERK kinase (MEK) and p38 kinase did not inhibit TLR2 mRNA
up-regulation by LPS. In contrast, LPS-mediated TLR2 mRNA induction was
abrogated by pretreatment with a high concentration of an NF-B
inhibitor, curcumin, suggesting that NF-B activation may be
essential for the process. Our results indicate that TLR2, in contrast
to TLR4, can be induced in macrophages in response to bacterial
infections and suggest that although TLR2 is dispensable for the
initial host responses against LPS, it may contribute to the
accelerated macrophage responses seen at subsequent stages of
infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant mouse IL-2, IL-1, TNF-, IFN-, and human
IL-15 were purchased from Peprotech (Seattle, WA). PD98059, a specific
inhibitor of ERK kinase (MEK), and SB208530, a specific inhibitor of
p38 kinase, were purchased from Calbiochem (San Diego, CA). LPS from
Escherichia coli serotype B6:026, curcumin, anisomycin, and
PMA were obtained from Sigma (St. Louis, MO). RPMI 1640 was from Life
Technologies (Rockville, MD). Synthetic E. coli-type lipid
A, compound 506, was previously described (28).
Anti-phospho-ERK mAb and anti-p38 MAP kinase polyclonal Ab were
obtained from New England Biolabs (Beverly, MA) and Santa Cruz
Biotechnology (Santa Cruz, CA), respectively.
Cells
All cell lines were grown in tissue culture flasks at 37°C in 5% CO2/95% air and passaged every 2 or 3 days to maintain logarithmic growth. A mouse macrophage cell line, RAW264.7, was obtained from The Institute of Physical and Chemical Research cell bank (Tsukuba, Japan) and maintained in DMEM with 10% FCS (Sigma).
Adherent cells from spleens of naive BALB/c, C3H/HeN, or C3H/HeJ mice were used as a source of mouse macrophages. Briefly, isolated splenocytes suspended in DMEM containing 10% FCS were cultured in plastic plates for 1 h at 37°C, nonadherent cells were removed, and the fresh complete medium was added to the adherent cells with or without stimulants.
Northern blot analysis
Total cellular RNAs were extracted using TRIZOL reagent (Life Technologies, Rockville, MD) according to the manufacturer’s instructions. Aliquots (20 µg) of the total RNAs were fractionated in a 1% agarose gel containing 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA (pH 7.0), and 6% (v/v) formaldehyde and transferred to a nylon membrane. After UV cross-linking, membranes were soaked in prehybridization solution (6x SSC, 5x Denhardt’s reagent, 0.5% SDS, 100 µg/ml denatured salmon sperm DNA, and 50% formamide) for 3 h at 42°C followed by incubation with 32P-labeled probe in hybridization solution (6x SSC, 0.5% SDS, 100 µg/ml denatured salmon sperm DNA, and 50% formamide) for 14 h at 42°C. The membranes were washed in 2x SSC, 0.1% SDS for 10 min twice at room temperature and in 0.1x SSC, 0.1% SDS for 10 min twice at 50°C and were exposed to Fuji RX-U films (Fuji Film, Tokyo, Japan).
For specific DNA probes, partial cDNA fragments containing the full
coding regions of mouse TLR2, TLR4, and -actin (including no 5' or
3' noncoding regions) were prepared by RT-PCR using total RNA from
LPS-treated RAW264.7 cells as template and labeled with
[-32P]dCTP (New England Nuclear, Boston,
MA). The specific activities for the labeled probes were 1.5–5.0
x 106 cpm/ng.
Extract preparation and immunoblotting
Cells were lysed in PLC lysis buffer (50 mM HEPES (pH 7.0), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM NaPPi, 1 mM Na3VO4, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) at 1 x 108 cells/ml. The lysates were separated in SDS-polyacrylamide gels and then electrotransferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked for 2 h in 5% nonfat milk-TBST (20 mM Tris-HCl (pH 7.6), 0.15 M sodium chloride, 0.1% Tween 20), incubated with primary Abs in TBST for 1 h, washed three times with TBST, and incubated for 1 h with HRP-conjugated anti-mouse or rabbit Ig (Amersham Pharmacia Biotech) diluted 1:5000 in 5% nonfat milk-TBST. After three washes in TBST, the blot was developed with the enhanced chemiluminescence system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.
In vitro kinase assay
The GST-ATF2(1-109) fusion protein was prepared as previously
described (29). Cells were lysed in PLC lysis buffer at
1 x 108 cells/ml. p38 kinase was
immunoprecipitated with anti-p38 kinase polyclonal Ab and protein
A-Sepharose beads (Amersham Pharmacia Biotech) from 100 µl lysate.
The beads were extensively washed with PLC lysis buffer three times
followed by kinase buffer (20 mM Tris-HCl (pH 7.4), 20 mM
MgCl2, and 2 mM EGTA) once. The beads were then
added with 40 µl kinase buffer containing 500 ng GST-ATF2(1-109) and
0.5 µCi [-32P]ATP (New England Nuclear)
and incubated at 30°C for 20 min. The phosphorylation of
GST-ATF2(1-109) was analyzed by SDS-PAGE and autoradiography.
Luciferase assays
RAW264.7 cells were transiently transfected with 2 µg of
pGL3-NF-B/Luc (a luciferase reporter construct containing consensus
NF-B binding sequence) and 0.2 µg of pRL/SV40 (an internal
control) by Lipofectamine (Life Technologies) according to the
manufacturer’s instructions. Twenty-four hours after the transfection,
some cells were pretreated with indicated chemicals for 30 min followed
by the addition of 1 ng/ml LPS. After 8-h incubation with LPS, cells
were lysed, and the luciferase activity was measured by using the
Dual-Luciferase Reporter Assay System (Toyo Ink, Tokyo, Japan)
according to the manufacturer’s instructions. Background luciferase
activity was subtracted, and the data are presented as means ± SD
of triplicate samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several laboratories including our own have recently reported that both TLR2 and TLR4 are capable of mediating LPS responsiveness (16, 17, 18, 19, 20). However, the transcriptional regulation of these two proteins in response to Gram-negative bacteria has not been fully elucidated. Thus, we investigated the gene expression of TLR2 and TLR4 in mouse macrophages that play essential roles in innate immunity.
Macrophages isolated from spleens of naive BALB/c mice were treated
with LPS or IL-15, a cytokine known to activate macrophages, for 2
h in vitro followed by total RNA preparation. In the Northern blot
analysis, TLR2 mRNA was weakly expressed in unstimulated macrophages
and significantly increased by LPS treatment (Fig. 1
A). Interestingly, IL-15, a
cytokine shown to play an important role in the early phase of
bacterial infection, also increased TLR2 mRNA level. In contrast, TLR4
mRNA expression remained constant after LPS or IL-15 treatment (Fig. 1A). Multiple mRNA bands were always detected using TLR4
probes probably due to alternative splicing.
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B,
splenic macrophages expressed significantly more TLR2 mRNA within
2 h after the LPS injection. The TLR2 mRNA level remained elevated
for at least 8 h after the LPS injection. In contrast, there was
no evident increase of TLR4 mRNA (Fig. 1B). Taken together,
these results indicate that the mRNA of two putative LPS receptors,
TLR2 and TLR4, are differently regulated in macrophages by
LPS. Regulation of TLR2 and TLR4 gene expression in mouse macrophage cell line, RAW264.7
RAW264.7 is a well-established mouse macrophage cell line. To
determine the effects of LPS and various cytokines on the TLR2 mRNA
expression, RAW264.7 cells were treated for 2 or 8 h with LPS,
IL-15, IL-2, IL-1, IFN-, or TNF-, and total RNAs were
isolated. All the cytokines examined increased the TLR2 mRNA level in
<2 h (Fig. 2A). The TLR2 mRNA
increases by LPS, IL-2, IL-1, IFN-, or TNF- were rather
transient, decreasing within 8 h of stimulation, whereas the
effect of IL-15 remained relatively longer. In contrast, there was no
significant increase of TLR4 mRNA by any of the treatments (Fig. 2A). Similar results were obtained from J774.1, another
well-established mouse macrophage cell line (data not shown). In a more
detailed time course experiment, TLR2 mRNA increased by 1 h,
peaked at 2 h, and decreased below the basal level by 12 h
after LPS stimulation (Fig. 2B). In contrast, TLR4 mRNA
remained constant for 12 h and decreased by 24 h
after LPS stimulation. TLR2 gene expression is also up-regulated in
RAW264.7 cells by synthetic lipid A in a concentration-dependent manner
(Fig. 2C), suggesting that the TLR2 mRNA increase is not due
to other substances possibly contaminated in the LPS and is mediated at
least partly by the lipid A region of LPS.
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A)
and various cytokines are secreted from macrophages by LPS treatment,
it is possible that LPS increases TLR2 mRNA indirectly by inducing
cytokine secretion. To rule out this possibility, RAW264.7 cells were
pretreated with a protein synthesis inhibitor, cycloheximide. As shown
in Fig. 2D, cycloheximide rather enhanced LPS-mediated
increase of TLR2 mRNA, suggesting that LPS directly increases TLR2 mRNA
in macrophages. The result also indicates that there may be a negative
feedback mechanism for TLR2 induction that requires new protein
synthesis. Additionally, cycloheximide pretreatment did not inhibit the
TLR2 mRNA increase by any of the examined cytokines (data not shown),
suggesting that their effects on TLR2 gene expression are also
direct.
Both TLR2 and TLR4 mediate LPS signals in in vitro studies.
To determine whether the LPS-induced TLR2 mRNA increase is mediated by
TLR4 or TLR2, we examined macrophages from C3H/HeJ mice that contain
nonfunctional mutation in the TLR4 gene. When isolated splenic
macrophages from this mouse strain were stimulated with synthetic lipid
A, TLR2 mRNA increase was severely impaired compared with that of
macrophages isolated from the closely related C3H/HeN mice (Fig. 2E). This finding suggests that TLR4 is essential for the
lipid A-induced TLR2 mRNA increase.
ERK and p38 kinase are not essential for the LPS-mediated TLR2 mRNA induction
LPS stimulation of macrophages is known to activate MAP kinase
pathways, including ERK, JNK, and p38 kinase (4, 5, 6, 7, 8, 9). To
investigate whether ERK and p38 kinase pathways are involved in mTLR2
mRNA up-regulation, RAW264.7 cells were pretreated with a specific
inhibitor of ERK (PD98059) or p38 kinase (SB208530) pathway followed by
LPS stimulation. Pretreatment with SB208530 only slightly inhibited the
mTLR2 mRNA increase even at 50 µM (Fig. 3A). Unexpectedly, PD98059
treatment enhanced TLR2 mRNA increase (Fig. 3A). To further
elucidate the role of MAP kinase pathways in TLR2 gene expression in
macrophages, we treated RAW264.7 cells with anisomycin, a potent
activator of JNK and p38 kinase (30). As shown in Fig. 3C, no increase was observed in the TLR2 mRNA level after
anisomycin treatment. Taken together, these findings suggest that
neither ERK nor p38 kinase pathway is essential for LPS-mediated
up-regulation of TLR2 mRNA. On the contrary, ERK activation may rather
inhibit TLR2 mRNA expression.
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B activation may regulate TLR2 mRNA up-regulation
Curcumin has recently been reported to inhibit JNK activation in
various cell types at a concentration of 5 or 10 µM
(31). Higher concentration of curcumin (50 µM or
higher), however, inhibits NF-B activation (31). When
RAW264.7 cells were treated with curcumin before LPS stimulation, it
inhibited LPS-mediated TLR2 mRNA up-regulation at 50 µM, whereas
lower concentrations of curcumin had no inhibitory effect (Fig. 4A). To investigate whether
this inhibitory effect correlated with the inhibition of NF-B
activity, we performed a luciferase reporter assay. RAW264.7 cells were
transfected with a luciferase reporter construct containing a NF-B
recognition sequence followed by the treatment with curcumin and LPS.
As shown in Fig. 4B, concentration-dependent inhibition of
NF-B activity by curcumin correlated well with the inhibition of
TLR2 gene expression, suggesting that NF-B activation may be
essential for the LPS-mediated TLR2 mRNA up-regulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The roles of TLR2 and TLR4 as LPS receptors are still controversial.
Although both human and mouse TLR2 were shown to mediate LPS signaling
in vitro (16, 17, 18), the role of TLR2 in LPS signaling in
vivo is questioned due to several recent findings. Studies examining a
single autosomal locus (lps) responsible for the LPS
hyporesponsiveness (lpsd) of two mouse
strains (C3H/HeJ and C57BL10/ScCr) (21, 22) found TLR4
gene in the lps locus and also identified mutations in the
TLR4 genes of both lpsd strains, strongly
suggesting that the defective TLR4 is responsible for the LPS
hyporesponsiveness of these mice. More recently, both TLR2- and
TLR4-lacking mice have been generated. Although TLR4-lacking mice show
LPS-hyporesponsiveness very similar to that of the
lpsd mouse strains (32),
macrophages from TLR2-lacking mice showed TNF- secretion similar to
that of wild-type mice in response to LPS stimulation
(23). These findings are consistent with the idea that
TLR4 is essential in LPS signaling, whereas TLR2 is dispensable.
Our current finding may explain the discrepancies between gene-lacking
mice and in vitro transfection data. In unstimulated macrophages, TLR4
mRNA is constitutively expressed, whereas TLR2 gene expression is
relatively low. Thus, TLR4 may be essential to initiate the innate
immunity in the first encounter with Gram-negative bacteria, whereas
TLR2 is induced later by TLR4 signals and becomes the second LPS
receptor to help the immune response against the bacteria. Consistent
with this hypothesis, the sensitivity of lpsd
mice is restored by treatment with IFN- or bacillus
Calmette-Guérin (33, 34, 35, 36). The expression
level of TLR2 in macrophages is likely to be higher after these
treatments in lpsd mice as TLR2 mRNA is
up-regulated by various cytokine stimulations in RAW264.7 cells (Fig. 2A) and in C3H/HeJ mouse macrophages (Fig. 2E).
It should be noted, however, that our findings are based on the
measurements of mRNA levels rather than protein levels. The surface
expression of TLR2 may not necessarily correlate with the mRNA levels,
and further studies are warranted on this matter.
Several other explanations are also possible. One possible explanation is that TLR2 and TLR4 respond to different types of LPS. Cells from C3H/HeJ mice are as sensitive as their normal counterparts to certain types of LPS (e.g., Porphyromonas gingivalis LPS) (37). There is divergence in LPS structure among Gram-negative bacteria, and it is reasonable to presume that TLR4 responds to certain types of LPS better than TLR2 while TLR2 responds better to others. A second possibility is that TLR2 and TLR4 may cooperate to respond to LPS. It is not known how TLRs are involved in the LPS receptor complex, and it is possible that TLR2 needs to associate with TLR4 to form a heterodimer or oligomer for proper functions, whereas TLR4 can work as a homodimer or oligomer. This seems inconsistent with the in vitro transfection data that the transfection of TLR2 alone can confer LPS responsiveness to cells (16, 17, 18). However, it cannot be excluded that the transfected TLR2 cooperates with the weakly expressed endogenous TLR4. Thirdly, it is possible that the published in vitro results that TLR2 mediates LPS signals are due to the nonphysiological TLR2 expression level, and TLR2 is not involved in LPS response in physiological conditions.
Lastly, it is also possible that TLR2 may have just responded to the other bacterial components contaminating the LPS used in the in vitro assays.
Recently, it has been reported that TLR2 mediates signals from
liparabinomannan, peptidoglycan, lipoproteins, and lipoteichoic acid
(24, 26, 27, 38, 39, 40). TLR2-defective mice showed
unresponsiveness to these components, suggesting that TLR2 is essential
for the signal transduction (23). It remains to be seen
whether these bacterial components also increase the TLR2 mRNA in
macrophages. However, because TNF- up-regulates TLR2 gene expression
and serum TNF- level increases in response to these components, the
same positive feedback mechanism should exist in response to bacteria
containing these components, and the increased TLR2 expression will
probably contribute to the accelerated immune responses by macrophages.
If so, regulation of TLR2 expression may be one of the
immune-regulatory mechanisms commonly involved in host defense against
many bacterial strains.
Although LPS is known to activate various MAP kinase pathways
(4, 5, 6, 7, 8, 9), the activation of ERK, p38 kinase, or JNK does not
seem to be essential for the induction of TLR2 gene expression in
RAW264.7 cells (Figs. 3 and 4). PD98059, a specific inhibitor of ERK
pathway, rather enhanced TLR2 mRNA increase by LPS, suggesting that ERK
activation has an inhibitory effect on TLR2 expression in macrophages.
Anisomycin, a potent activator of both JNK and p38 MAP kinase, fail to
induce TLR2 mRNA, suggesting that these MAP kinases are not sufficient
for TLR2 gene up-regulation. Additionally, SB208530, a specific
inhibitor of the p38 MAP kinase pathway, was only slightly inhibitory
to LPS-mediated TLR2 induction. These results were rather unexpected,
because they were in sharp contrast to those with T cells. We have
previously reported that in T cells PMA potently increased TLR2 mRNA
expression and both PD98059 and SB208530 effectively inhibited the
increase (18). This difference clearly suggests that the
regulatory roles of MAP kinase activation pathways in the regulation of
TLR2 mRNA vary considerably in different cell types.
Interestingly, ERK and p38 kinase pathways were reported to play
different roles in the expression of some LPS-responsive genes in
macrophages. For example, P38 kinase is essential for NO synthase
induction, whereas ERK plays only a minor role (41). Also,
while p38 kinase promotes IL-12 (p40) expression, ERK inhibits it
(41). It is of note that LPS-mediated NF-B activation
is known to play a major role in the induction of both genes (42, 43). Interestingly, phosphoglycan, a major cell wall protein of
Leishmania, was suggested to suppress immune response by
decreasing IL-12 production via the activation of ERK pathway
(41), and this mechanism can be shared by other pathogens.
Our present findings suggest that the suppression of TLR2 expression
through ERK activation may be another mechanism for
Leishmania to escape immune response.
Instead, in macrophages, the NF-B activation seems to be essential
for the LPS-mediated TLR2 induction. LPS is a strong activator of
NF-B (2, 3), and all of the cytokines shown here to
increase TLR2 expression are known activators of NF-B
(44). Additionally, curcumin, an inhibitor of NF-B
activity at high concentrations, completely inhibited LPS-mediated TLR2
induction at 50 µM (Fig. 4). Although curcumin is not a specific
inhibitor of NF-B activation, the dose response of TLR2 mRNA
inhibition correlated well with that of NF-B activation. From these
observations it is likely that LPS-induced TLR2 gene expression is
mediated by NF-B activation. In agreement with this hypothesis,
there are NF-B recognition sites in the promoter region of mouse
TLR2 gene (T. Musikacharoen, manuscript in preparation).
While this paper was being reviewed, Medvedev et al. also reported the
rapid increase of TLR2 mRNA in LPS-treated mouse macrophages
(45). In their report, however, TLR2 mRNA elevation lasted
for at least 12 h, longer than our finding with RAW264.7 cells
(Fig. 2B). The longer duration of TLR2 mRNA could be due to
the effects by a small number of contaminating other types of cells,
such as T cells. In fact, we also found longer TLR2 mRNA elevation in
mouse macrophages than in macrophage cell lines after LPS treatment
(data not shown).
In contrast to our results, TLR2 mRNA did not seem to increase in human monocytes after 3-h treatment with LPS (46). The reasons for this discrepancy are not clear. It is possible that TLR2 gene expression is differently regulated in monocytes and in tissue macrophages. Alternatively, it is also possible that the regulation varies among different species.
Recent reports have indicated that TLR4 mRNA expression in macrophages
was decreased within a few hours of LPS treatment (45, 47), whereas we could not observe the obvious TLR4 mRNA decrease
until 24 h after the LPS treatment (Fig. 2B). In
another report, Muzio et al. (48) found that LPS increased
TLR4 mRNA in monocytes and polymorphonuclear leukocytes. While
performing the time course experiments using RAW264.7 cells, we also
observed a slight decrease or increase of TLR4 mRNA at 2 or 4 h of
LPS treatment on several occasions. In most experiments, however, TLR4
mRNA remained constant. Thus, the discrepancies between their results
and ours are most likely due to the slight differences of stimulation
conditions (cell densities, etc.).
In summary, this study implies that the expression of the two putative
LPS signaling receptor genes, TLR2 and TLR4, are differently regulated
in mouse macrophages. TLR4 was constitutively expressed and remained
constant after various stimulations, including LPS. In contrast, gene
expression of TLR2 significantly increased after LPS treatment both in
vitro and in vivo. This led us to suggest a model shown in Fig. 5. When Gram-negative bacteria invade the
host, macrophages first recognize LPS through the constitutively
expressed TLR4. Later, TLR2 is induced directly by LPS or indirectly
through secondary cytokines. Through the newly synthesized TLR2,
macrophages respond better to LPS or other bacterial components such as
lipoproteins that are membranous components of both Gram-positive and
Gram-negative bacteria. There are six mammalian TLR proteins that have
been identified so far, and the number will probably increase in the
future. It remains to be seen whether this inducibility is unique to
TLR2 or is shared by some other members of TLRs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tetsuya Matsuguchi, Laboratory of Host Defense and Germfree Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
3 Abbreviations used in this paper: ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; TLR, Toll-like receptor; MAP, mitogen-activated protein; MEK, MAP/ERK kinase.
Received for publication March 6, 2000. Accepted for publication August 17, 2000.
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