* The Pulmonary Center, Boston University School of Medicine, Boston Massachusetts, and
Mycobacteria Research Laboratories, Department of Microbiology, Colorado State University, Ft. Collins, Colorado
Correspondence: Dr. Matthew J. Fenton, Pulmonary Center R-220, Boston University School of Medicine, 80 East Concord St., Boston MA 02118-2394. E-mail: mfenton{at}bu.edu
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B, activator protein-1 (AP-1), and mitogen-activated protein
(MAP) kinases. These TLR agonists induced similar levels of NF-B and
AP-1 DNA-binding activity, as well as trans-activation
function. Unexpectedly, these TLR agonists induced tumor necrosis
factor 
secretion, whereas only LPS was capable of inducing
interleukin-1ß and nitric oxide secretion. Thus, different TLR
proteins are still capable of activating distinct cellular responses,
in spite of their shared capacities to activate NF-B, AP-1, and MAP
kinases.
Key Words: signal transduction • tuberculosis • innate immunity
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Interleukin (IL)-1ß, IL-6, and
IL-12], coactivator molecules (B7.1), and nitric oxide (NO)
[6
, 7
]. Currently, nine distinct TLR
proteins have been identified [8
9
10
11
], although the
specific roles that these receptors play in regulating host innate
immune responses remain poorly understood.
Different TLR proteins recognize a variety of chemically diverse
bacterial products. LPS is predominantly recognized by TLR4
[4
, 12
, 13
], whereas LAM,
peptidoglycan, and bacterial lipoproteins are recognized by TLR2
[7
, 14
15
16
]. Although it is clear that
purified bacterial products can initiate TLR-dependent signaling, the
relative contributions of different TLR proteins, as well as
TLR-independent signaling, to cellular responses induced by whole
bacteria have only recently been examined. Takeuchi et al. reported
that TLR4-deficient murine peritoneal macrophages exhibit normal
production of TNF- and IL-6 after stimulation with heat-killed
Staphylococcus aureus in vitro, compared with that of
wild-type cells [17
]. In contrast, TLR2-deficient
macrophages exhibited a moderate reduction in S.
aureus-induced cytokine production compared with that of wild-type
cells. We recently compared the in vitro responses of normal and
TLR-deficient macrophages to live Mycobacterium tuberculosis
bacilli. These studies revealed that TNF- production is largely
dependent on cellular activation via TLR4, whereas M.
tuberculosis-induced NO production is independent of TLR proteins
[18
]. Although cellular responses to purified bacterial
products often provide little insight into responses initiated by
challenge with whole bacteria, purified TLR agonists are still
essential tools for dissecting the signal transduction pathways that
mediate TLR-dependent cellular responses.
TLR proteins are members of a larger family of receptors, which
includes the type I IL-1 receptor, the IL-18 receptor, and the
cytosolic adapter protein (AP) MyD88 [19
]. The
engagement of some TLR proteins by bacterial products sometimes
requires coreceptors, such as CD14 and MD-2 [20
,
21
]. Intracellular signaling triggered by engagement of
the IL-1 receptor, the IL-18 receptor, and the TLR proteins involves a
shared cascade of APs and kinases [reviewed in ref. 22
]. These
include MyD88, IL-1 receptor-associated kinase (IRAK), TNF
receptor-associated factor 6 (TRAF6), and several mitogen-activated
protein (MAP) kinase kinase kinases. This signaling pathway ultimately
leads to the activation of transcription nuclear factor (NF)-B and
phosphorylation of MAP kinases, although these events are not the only
consequences of TLR-mediated signaling. For example, both IL-1ß and
LPS have been reported to activate several additional protein kinases
[23
24
25
], lipid kinases [26
,
27
], and phospholipases [28
,
29
]. To date, no studies have directly compared the
signaling pathways activated by different TLR agonists.
We have now compared the capacities of different TLR agonists to trigger several signal transduction pathways and ultimately to activate selected cellular responses. Here we report that two mycobacterial TLR2 agonists, soluble tuberculosis factor (STF) and LAM, induce a different pattern of cellular responses than that induced by the TLR4 agonist LPS.
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In experiments using 3E10 cells, which contain a stably transfected
CD25 reporter gene under the control of the NF-B-dependent ELAM-1
promoter, CD25 expression was measured by flow cytometry as previously
described [14
]. Data were collected using CellQuest
software (Becton Dickinson, Bedford, MA) and expressed as the ratio
(fold activation) of the percent of CD25+ cells in
unstimulated and stimulated cell populations (gated to exclude the
lowest 5% of cells based on mean fluorescence). The 95% confidence
limit for nonspecific fluorescence was established using isotype
control antibodies. Fluorescein isothiocyanate and
phosphatidylethanolamine-conjugated anti-human CD25 monoclonal
antibodies were purchased from Becton Dickinson.
LPS (purified from Eschericia coli 055:B5) was purchased from Sigma (St. Louis, MO) and repurified by the method of Hirschfeld et al. [31 ]. Mycobacterial arabinose-capped LAM, purified from a rapidly growing avirulent Mycobacterium species, and mannosylated phosphatidylinositol (PIM) were purified as previously described [32 , 33 ]. The levels of contaminating LPS in the LAM and PIM preparations were determined using a quantitative Limulus lysate assay (BioWhittaker) and were <1-pg/mL final concentrations in all experiments.
Preparation of mycobacterial STF and culture filtrate factors
M. tuberculosis strain H37Ra (ATCC 25177) was
purchased from the ATCC. Bacterial cultures were grown in LPS-free
Middlebrook 7H9 medium supplemented with Tween 80 and
albumin-deoxycholate medium supplement (Difco, Detroit, MI) at 37°C
in LPS-free flasks under biosafety-level-3 conditions to
mid-logarithmic phase [optical density at 620 nm, 0.4). Bacteria were
then removed by centrifugation followed by two rounds of filtration
through a 0.22-µm-pore-size membrane. This short-term culture
filtrate was digested with proteinase K (100 µg/mL) for 18 h at
56°C and then used as a crude source of STF. Further purification of
STF was accomplished by extraction with Triton X-114 as previously
described [34
]. Triton X-114 was added to crude ice-cold
STF preparations until a final concentration of 4% was achieved. This
mixture was incubated overnight at 4°C on a rotating wheel and then
warmed to 37°C to promote the formation of two phases. The lower
phase containing the Triton X-114 and glycolipids was removed, and the
glycolipids were recovered by acetone precipitation. Culture filtrates
were also subjected to size fractionation using preparative sodium
dodecyl sulfate (SDS)-15% polyacrylamide gels [35
]. The
resolved products were recovered by electroelution using a Whole Gel
Eluter (Bio-Rad Laboratories, Hercules, CA) [35
].
Individual fractions were lyophilized, dissolved in LPS-free
phosphate-buffered saline, and adjusted to 1 µg of total protein per
µL with phosphate-buffered saline.
Measurement of cytokine and NO levels
TNF- and IL-1ß protein levels in culture supernatants were
determined using a specific enzyme-linked immunosorbent assay (ELISA)
(R&D Systems, Minneapolis, MN), as recommended by the manufacturer.
Levels of NO catabolite nitrite in the culture supernatants were
measured using the Griess reagent assay as previously described
[36
]. All assays were performed in triplicate, and data
are expressed as mean values ± SD. The data were
subsequently analyzed using an analysis of variance to determine
statistical significance.
Measurement of TNF-, IL-1ß, and NOS2 mRNA levels by reverse
transcription-PCR
Total RNA from RAW 264.7 murine macrophages was purified using
RNeasy (Qiagen, Valencia, CA) as recommended by the manufacturer.
Reverse transcriptase (RT) reactions to generate cDNA were performed
using AMV RT (Promega, Madison, WI). PCR was performed using 0.5 µg
of cDNA, 0.12 µM oligonucleotide primers (each), 2 mM
MgCl2, 0.2 mM deoxynucleotide triphosphatase, and 1 U of
Taq polymerase (Promega) in a final reaction volume of 25
µL. Thirty amplification cycles were performed (each cycle consisting
of 1 min of 95°C denaturation, 1 min of 55°C annealing, and 1 min
of 72°C extension). PCR primers used in this study are listed below
and were purchased from Gibco-BRL (Frederick, MD): sense strand
inducible nitric oxide synthase (iNOS) primer, 5'-AAT GGC AAC ATC AGG
TCG GCC ATC ACT-3'; anti-sense strand iNOS primer, 5'-GCT GTG TGT CAC
AGA AGT CTC GAA CTC-3'; sense strand TNF- primer, 5'-ATG AGC ACA GAA
AGC ATG ATC-3'; anti-sense strand TNF- primer, 5'-TAC AGG CTT GTC
ACT CGA ATT-3'; sense strand IL-1ß primer, 5'-TAC AGG CTC CGA GAT GAA
CAA CAA-3'; anti-sense strand IL-1ß primer, 5'-TGG GGA AGG CAT TAG
AAA CAG TCC-3'; sense-strand ß actin primer, 5'-TCA TGA AGT GTG ACG
TTG ACA TCC GT-3'; anti-sense strand ß actin primer, 5'-CCT AGA AGC
ATT TGC GGT GCA CGA TG-3'. As a control for contaminating genomic DNA,
parallel PCR reactions were performed in which the template nucleic
acids were not reverse transcribed. After amplification, portions of
the PCR reactions were electrophoresed on a 2% agarose gel and
visualized using ethidium bromide.
Western blot analysis
Whole-cell lysates were prepared from unstimulated RAW 264.7
macrophages, and from cells stimulated with different TLR agonists for
various times as we have previously described [37
].
Aliquots containing 20–100 µg of total protein per lane were
fractionated on SDS-12% polyacrylamide gels and then
electrophoretically transferred to nitrocellulose membranes (Bio-Rad).
Activated MAP kinases were detected using specific antisera against the
phosphorylated forms of extracellular regulated kinase (ERK) and c-Jun
kinase (JNK) (New England Biolabs, Beverly, MA), according to the
manufacturer’s instructions. Membranes were developed using a donkey
anti-rabbit antiserum linked to horseradish peroxidase (Amersham
Pharmacia Biotech, Piscataway, NJ) and then visualized using an
enhanced chemiluminesence reagent (chemiluminescence-horseradish
peroxidase substrate system; Pierce Corp., Rockford, IL).
Electrophoretic mobility shift assays
Nuclear extracts were prepared from RAW 264.7 cells as described
by Schreiber et al. [38
] and then analyzed as we have
previously described [14
]. Double-stranded
oligonucleotides containing a single consensus NF-B (5'-AGT TGA
GGG GAC TTT CCC AGG C-3')- or AP-1 (5'-CGC TTG ATG
ACT CAG CCG GAA-3')-binding site were used as electrophoretic
mobility shift assay (EMSA) probes. DNA probes were labeled with
-32P-labeled deoxynucleoside triphosphates (DuPont-NEN,
Boston, MA) using E. coli DNA polymerase Klenow fragment
(U.S. Biochemicals, Cleveland, OH), and unincorporated nucleotides were
removed using Sephadex G-25 columns (5 Prime-3 Prime Inc., Boulder,
CO). Nuclear extracts (typically 3 µg) were incubated with
radiolabeled-probe DNA in the presence of 2 µg of poly dI-dC
(Pharmacia), 1.0 mM ethylenediaminetetraacetate, 10 mM Tris-HCl (pH
7.9), 25 mM glycerol, and 0.5 mM dithiothreitol in a final volume of 20
µL. Binding reactions were then incubated at room temperature for 30
min. After incubation, a portion of each binding reaction was loaded
onto 7% nondenaturing low-ionic-strength polyacrylamide gel. The gels
were then dried and visualized by autoradiography.
Plasmids
The NF-B-dependent ELAM-luciferase reporter plasmid was
obtained from Douglas Golenbock and was previously described
[30
]. The AP-1-luciferase reporter plasmid, containing
four copies of a consensus AP-1 site, was previously described
[39
]. All plasmids were prepared using Qiagen plasmid
DNA purification columns, DNA was eluted from the columns using
LPS-free buffers, and contaminating LPS levels were found to be less
than 10 pg/mL. Furthermore, all plasmid preparations were unable to
activate the LPS-sensitive RAW 264.7 macrophages at the concentrations
used for transfection, demonstrating that the plasmids were not
contaminated with LPS (data not shown).
Transfection and reporter assays
Transient transfections were performed using Super-Fect reagent
(Qiagen) according to the manufacturer’s instructions, with minor
modifications that we previously described [14
].
Transfection mixtures containing a total of 3 µg of plasmid DNA were
incubated with the RAW 264.7 cells for 2–3 h, whereupon the reaction
was removed from the cells, and fresh medium containing serum was
added. On the following day, individual wells were left untreated or
were stimulated with TLR agonists for various times as indicated in the
figures. Cells were then incubated for an additional 5–18 h prior to
harvesting. Luciferase assays were performed using the Luciferase Assay
System (Promega), according to manufacturer’s instructions. All
transfection experiments were performed in triplicate, repeated at
least three times using different plasmid preparations, and a single
representative experiment is shown. Each single experiment represents
triplicate independent transfections, and data are expressed as average
values ± SD.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 View larger version (16K): [in a new window] |
Figure 1. PIM is a TLR2 agonist that is present in STF. Size-fractionated
mycobacterial filtrate fractions were prepared as described in
Materials and Methods and tested for TLR2 agonist activity using
3E10/TLR2 cells. Aliquots of size-fractionated STF were added to
3E10/TLR2 cells at a final concentration of 20 µg/mL. Cells were also
stimulated with crude STF (20 µL/mL) as a positive control.
Twenty-four hours later, the cells were stained with a fluorescein
isothiocyanate-labeled anti-CD25 monoclonal antibody and subjected to
flow cytometry analysis to measure the expression of the CD25. Data are
expressed as the ratio (fold activation) of the percents of
CD25+ cells in unstimulated and stimulated cell populations
that were gated to exclude the lowest 95% of FL1 fluorescence obtained
using the isotype control antibody (A). 3E10/TLR2 cells were stimulated
with STF (20 µL/mL), purified PIM (1 µg/mL), and purified AraLAM (1
µg/mL), and then analyzed 24 h later as described in the text
(B). STF was extracted with Triton X-114 to yield Triton-soluble and
aqueous STF fractions as described in Materials and Methods. The
ability of these fractions and crude STF to activate 3E10/TLR2 cells
was analyzed as described in the text (C). In B and C, assays were
performed in triplicate, and repeated on three separate occasions. A
single representative experiment is shown, and the data are expressed
as mean values ± SE.
|
B-dependent promoter (3E10 cells), as well as lines that
overexpress TLR2 (3E10/TLR2) and TLR4 (3E10/TLR4). As shown in Figure 1B
, purified PIM was capable of activating the 3E10/TLR2 cells. PIM
could not activate the 3E10 or 3E10/TLR4 cells (data not shown),
demonstrating that PIM is a specific TLR2 agonist. Because PIM can be
extracted from aqueous solutions using Triton X-114
[34
], we then sought to determine whether the TLR2
agonist activity could also be extracted from crude STF with Triton
X-114. As shown in Figure 1C , the TLR2 agonist activity could be
completely removed from crude STF by Triton X-114 extraction (aqueous
STF) and subsequently recovered from the Triton-soluble layer
(Triton-soluble STF). This finding also suggests that additional minor
TLR2 agonists are present in STF (e.g. fractions 2 and 13) and are
lipid conjugates such as glycolipids or lipoproteins. Last, analysis of
this Triton-extracted STF using thin-layer chromatography revealed the
presence of two major species that shared identical mobilities with
monophosphatidylinositol and PIM (PIM1 and
PIM2, respectively; data not shown). It should be noted
that PIM1 and PIM2 have similar mobilities on
SDS-PAGE (
6 kDa) and thus are not likely to be the TLR2 agonists
contained in fractions 2 and 13 (Fig. 1A) . Taken together, these data
indicate that the mycobacterial glycolipid PIM is a TLR2 agonist and is
likely to contribute to the total TLR2 agonist activity present in STF.
Our findings do not exclude the possibility that STF and even a single
size fraction purified from crude STF contain multiple TLR2 agonists.
LPS, LAM, and STF induce distinct cellular responses in macrophages
We subsequently sought to compare the cellular responses induced
by these various TLR2 and TLR4 agonists. RAW 264.7 murine macrophages
were stimulated with LPS, LAM, and STF for 24 h, and culture
supernatants were then collected. The levels of cytokines (TNF- and
IL-1ß) and nitrite, a stable catabolite of NO, were measured by ELISA
and the Greiss reagent assay, respectively. As shown in Figure 2
, LPS was capable of inducing RAW 264.7 macrophages to secrete
TNF-, IL-1ß, and NO. In contrast, LAM and STF were capable of
inducing only TNF- secretion. Higher concentrations of LAM, PIM, and
STF failed to induce IL-1ß and NO secretion (data not shown). Thus,
the cellular responses of macrophages induced by stimulation with these
TLR2 and TLR4 agonists were qualitatively distinct.
 View larger version (13K): [in a new window] |
Figure 2. Macrophage responses induced by LPS differ from those induced by
AraLAM, PIM, and STF. RAW 264.7 macrophages (15 x 105
cells/mL) were stimulated for 24 h with LPS (100 ng/mL), STF (10
µL/mL), PIM (1 µg/mL), or AraLAM (1 µg/mL). Levels of TNF- and
IL-1ß protein in the supernatants were then measured by ELISA.
Nitrite levels were also measured in these supernatants using the
Greiss reagent assay. All assays were performed in triplicate and
repeated on four separate occasions. A single representative experiment
is shown, and the data are expressed as mean values ±
SEM.
|
mRNA was constitutively expressed by the RAW 264.7 cells.
Previous studies have reported that the TNF- gene is constitutively
transcribed in unstimulated RAW 264.7 cells, although this mRNA is not
translated in the absence of stimulation [42
]. As
expected, LPS stimulated the expression of both IL-1ß and iNOS mRNA
in the RAW 264.7 cells. Similarly, LAM and STF induced IL-1ß mRNA
expression in the macrophages but failed to induce substantial iNOS
mRNA expression.
 View larger version (59K): [in a new window] |
Figure 3. AraLAM, PIM, and STF induce TNF- and IL-1ß but not iNOS
transcription in RAW 264.7 macrophages. RAW 264.7 macrophages were
stimulated with LPS (100 ng/mL), STF (10 µL/mL), PIM (4 µg/mL), or
AraLAM (4 µg/mL) for 4 h. Total RNA was then prepared from the
cells and analyzed by semiquantitative RT-PCR for iNOS, IL-1ß, and
TNF- mRNA as described in Material and Methods. PCR products were
fractionated on a 2% agarose gel and visualized using ethidium
bromide. PCR reactions that did not include RT RNA failed to generate a
product (data not shown), demonstrating the absence of contaminating
genomic DNA in the RNA preparations.
|
 View larger version (25K): [in a new window] |
Figure 4. AraLAM, PIM, and STF fail to induce translation of IL-1ß mRNA in RAW
264.7 macrophages. RAW 264.7 macrophages were stimulated for 6, 12, and
24 h with LPS (100 ng/mL), STF (10 µL/mL), PIM (1 µg/mL), or
AraLAM (1 µg/mL). Culture supernatants were then collected and saved.
Adherent macrophages were removed by scraping in ice-cold sterile
phosphate-buffered saline, freeze-thawed three times, and centrifuged
at 14,000 g for 10 min. These cell-free supernatants were
recovered and used as a source of intracellular lysate. IL-1ß protein
levels in the lysates and culture supernatants were measured by ELISA.
These assays were performed in triplicate and repeated on four separate
occasions. A single representative experiment is shown, and the data
are expressed as mean values ± SEM.
|
secretion, as
measured by ELISA (Figure 5A
). Using these concentrations, the levels of activated ERK and JNK
were measured by Western blotting using antibodies that specifically
recognize the phosphorylated forms of these MAP kinases. Antibodies
that recognize both phosphorylated and nonphosphorylated MAP kinases
were used as controls for equal loading. As shown in Figure 5B
, LPS was
a potent activator of both ERK and JNK. Multiple isoforms of ERK (p42
and p44) and JNK (JNK1 and JNK2) were detected and became
phosphorylated within 15 min after LPS stimulation. The TLR2 agonists
STF, PIM, and LAM also induced ERK phosphorylation, with maximal
activation observed within 35 min after stimulation. In contrast, these
TLR2 agonists were markedly less potent activators of JNK compared with
LPS. These TLR2 agonists were not wholly incapable of activating JNK,
as demonstrated by the finding that higher concentrations of these
agonists did activate this MAP kinase (Figure 5C)
. Even at higher
concentrations, the TLR2 agonists were poor inducers of JNK2
phosphorylation compared with LPS (Figure 5C)
. These studies indicate
that LPS, STF, LAM, and PIM induce similar levels of ERK activation in
the macrophages, whereas they differ in their capacities to activate
JNK in these cells.
 View larger version (51K): [in a new window] |
Figure 5. Differential activation of MAP kinase in RAW 264.7 cells by LPS and
mycobacterial glycolipids. RAW 264.7 cells were stimulated for 6 h
using the indicated amounts of LPS, STF, PIM, and AraLAM.
Secreted TNF- levels were then measured by ELISA (A). RAW 264.7
cells were stimulated for various times with amounts of LPS, STF,
PIM, and AraLAM that induced similar levels of TNF
secretion. After stimulation, cell lysates were prepared, fractionated
by SDS-PAGE (100 µg/lane for JNK and 20 µg/lane for ERK), and
probed with antibodies that specifically recognize the phosphorylated
forms of JNK and ERK (B). Membranes were subsequently stripped and
reprobed with antibodies that bound both phosphorylated and
nonphosphorylated forms of JNK and ERK. Arrows indicate the positions
of various JNK and ERK isoforms, based on published data
[29
]. RAW 264.7 cells were stimulated for 28 min with
the indicated concentrations of LPS, STF, and AraLAM. Cell lysates were
then prepared, fractionated on SDS-PAGE (100 µg total protein/lane),
and probed for JNK as described above (C).
|
B DNA-binding
activitiesB DNA-binding activity by
macrophages stimulated with LPS versus STF. As shown in Figure 6
,
NF-B DNA-binding activity was observed in nuclear extracts prepared
from macrophages stimulated with either LPS or STF. Two major
DNA-protein complexes were observed, and the specificity of these
complexes was demonstrated by the ability of excess unlabeled probe DNA
to compete for complex formation, whereas a nonspecific DNA probe could
not compete for complex formation. Like AP-1, both LPS and STF rapidly
induced similar levels of NF-B DNA-binding activity.
 View larger version (86K): [in a new window] |
Figure 6. NF-B and AP-1 activation by LPS and STF. RAW 264.7 macrophages
were stimulated with LPS (100 ng/mL) or STF (10 µl/mL) for 0.5, 1, 2,
or 4 h. Nuclear extracts from stimulated and unstimulated cells
were prepared as described in Materials and Methods, and then assayed
for AP-1 (A) or NF-B (B) DNA-binding activities by EMSA. The
positions of specific DNA-protein complexes containing AP-1 and NF-B
are indicated by arrows.
|
B-dependent
promoter activationB-dependent promoter activation. Luciferase reporter
plasmids containing AP-1- and NF-B-dependent promoters were
transiently transfected into RAW 264.7 cells, and a portion of the
cells was then stimulated with either LPS or STF for 4 h. Both
unstimulated and stimulated transfected cells were harvested, and
luciferase activity was measured as we previously described
[14
]. As shown in Figure 7
, similar levels of inducible AP-1- and NF-B-dependent reporter
gene activities were observed when transfected cells were stimulated
with either LPS or STF. Similar results were obtained when transfected
cells were stimulated with purified PIM (data not shown). Thus, like
the DNA-binding activities reported above, LPS and STF activated
similar levels of AP-1- and NF-B-dependent transcription, even
though these TLR agonists differed in their capacities to activate JNK
(see Fig. 5B
).
 View larger version (25K): [in a new window] |
Figure 7. NF-B and AP-1 luciferase reporter gene activation by LPS and STF.
RAW 264.7 macrophages were transiently transfected with one of the
following luciferase reporters: an NF-B-dependent ELAM-Luc promoter
(A) or a synthetic AP-1-Luc promoter (B). A portion of the cells was
then stimulated with LPS (100 ng/mL) or STF (10 µl/mL) for 2 or
4 h. Cells were then harvested, and luciferase activity was
measured as described in Materials and Methods. All transfection
experiments were performed in triplicate and repeated three times using
different plasmid preparations, and a single representative experiment
is shown. Data are expressed as average luciferase values from a single
experiment (subtracted for background) ± SD.
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secretion by the RAW
264.7 murine macrophage cell line. Unlike the TLR4 agonist LPS, these
glycolipid TLR2 agonists failed to induce IL-1ß and NO secretion by
these macrophages. Although LAM, STF, and PIM failed to induce iNOS
transcription, these TLR2 agonists were fully capable of inducing de
novo IL-1ß gene expression. In spite of the presence of abundant
IL-1ß mRNA in the LAM-, STF-, and PIM-stimulated cells, there was no
detectable intracellular IL-1ß protein, suggesting that the newly
transcribed mRNA was not being translated. These findings suggest that
LPS and the glycolipid TLR2 agonists trigger nonredundant signal
transduction pathways and that only LPS is capable of triggering the
pathway(s) necessary to activate IL-1ß mRNA translation. We
subsequently attempted to identify differences in intracellular
signaling in macrophages stimulated with LPS, LAM, STF, and PIM. These
studies revealed that LPS was a more potent activator of JNK compared
with LAM, STF, and PIM. In contrast, all of these TLR agonists were
capable of activating similar levels of ERK phosphorylation. In spite
of relative quantitative differences in the levels of JNK
phosphorylation induced by the different TLR agonists, there were no
significant differences in the levels of AP-1 DNA-binding activity and
trans-activation function observed in the stimulated RAW
264.7 macrophages. Moreover, NF-B DNA-binding activity and
trans-activation function were also found to be similar in
macrophages stimulated with LPS and STF, with similar results having
been obtained using LAM and PIM (data not shown). Thus, it is unlikely
that differences in the relative capacities of these TLR agonists to
induce JNK phosphorylation are sufficient to explain the inability of
LAM and STF/PIM to induce IL-1ß and NO secretion.
To date, a number of published studies have described a variety of
cellular responses that can be induced by different purified TLR
agonists [reviewed in ref. 1
22
44
]. All known TLR agonists have
been shown to induce TNF- secretion in monocytic cells
[14
, 45
46
47
48
]. Similarly, both TLR2 and TLR4
agonists have been previously reported to trigger rapid NF-B and MAP
kinase activation. Here, we have provided some of the first evidence to
demonstrate that, in spite of their similarities, different TLR
agonists can stimulate distinct cellular responses in macrophages. Very
recent studies by Hirschfeld et al. [49
] have reported
similar differences in cellular responses using primary murine
macrophages stimulated in vitro with LPS versus the TLR2 agonist
Porphyromonas gingivalis LPS.
It should be noted that the inability of the glycolipid TLR2 agonists
to induce IL-1ß and NO secretion is a characteristic that is not
shared by all TLR2 agonists. For example, IL-1ß secretion can be
induced in macrophages stimulated with a synthetic mycoplasmal
lipopeptide, a TLR2 agonist [50
]. Similarly, NO
production can be induced in macrophages by peptidoglycan
[2
], mycoplasmal lipopeptides [3
], when
cells were primed with IFN-
and Treponema glycolipids
[47
]. Also, Brightbill et al. [7
]
reported that the Borellia OspA and M.
tuberculosis 19-kDa lipoproteins could activate a reporter gene
under control of the iNOS promoter in transiently transfected RAW 264.7
cells [7
]. Using this same iNOS reporter plasmid, we
found that STF could activate reporter gene expression in transiently
transfected RAW 264.7 cells [18
], even though the
endogenous iNOS gene was not expressed (see Fig. 3
). Thus, care must be
taken to assure that reporter gene responses accurately reflect
endogenous gene expression.
It is reasonable to assume that the differential induction of IL-1ß
and NO by LPS and the glycolipid TLR2 agonists is the result of
differential intracellular signaling triggered by these stimuli. If
JNK-, AP-1-, and NF-B-dependent responses are not responsible for
these functional differences, then additional signaling pathways must
be considered. Activation of the TLR signal transduction pathway
involves sequential recruitment of MyD88, IRAK, and TRAF6
[51
52
53
]. This signaling pathway is largely shared by
the IL-1 and IL-18 receptors [reviewed in ref. 54
]. The intermediate
steps between TRAF6 and the IB kinase complex have not been clearly
identified, although several investigators have proposed that pathways
leading to MAP kinase activation are initiated at this level in the
signaling cascade [51
, 55
,
56
]. Earlier studies using LPS and IL-1ß have
implicated several additional protein and lipid kinases in signaling
initiated by TLR and IL-1 receptor proteins. These include Src family
protein tyrosine kinases, protein kinase C 
[29
],
phosphatidylinositol 3' kinase [27
, 57
],
and phosphatidylcholine-specific phospholipase C [28
].
Additional studies will be needed to determine whether the differential
capacities of LPS and the glycolipid TLR2 agonists to induce IL-1ß
and NO secretion arise from differences in the activation of any of
these parallel signaling pathways.
During the course of our studies, we obtained evidence demonstrating
that PIM is a TLR2 agonist and that it is likely to be present in crude
STF. PIM is a biosynthetic precursor of the larger glycolipid LAM and
may be one of the simplest structures known to have TLR agonist
activity. We are also unaware of any other studies demonstrating that
PIM is present in short-term culture filtrates from live M.
tuberculosis cultures. Recently, glycolipids with putative TLR2
agonist activity have been isolated from culture filtrates of
Treponema spirochetes [47
]. Thus, glycolipids
with TLR agonist activity may be secreted by pathogens present in vivo
at the sites of infection. These glycolipids are likely to be
immunomodulatory and act of neighboring uninfected cells in a paracrine
manner. This is consistent with previous studies showing that LAM can
be exported from M. tuberculosis-infected cells
[58
] and that exogenous LAM can be passively inserted
into the plasma membranes of uninfected cells [59
]. It
is tempting to speculate that glycolipids or other bacterial products
secreted by pathogens might be capable of affecting the development of
an acquired immune response. Support for this possibility comes from
recent studies by Mokuno et al. [60
] showing that
infection of mice with E. coli induces the recruitment of
/
T cells to the site of infection. These T cells express TLR2
but not TLR4 mRNA and can be directly activated by bacterial products
in vitro in a TLR2-dependent manner. If, like macrophages, T cells also
differ in their response to different TLR agonists, it is possible that
these agonists could selectively skew the development of normal Th1 and
Th2 responses. In conclusion, TLR2 agonists released by pathogens in
vivo are likely to affect the function of both early innate and
acquired immune responses.
Received December 18, 2000; revised January 25, 2001; accepted January 26, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 J. Lee, T.-H. Chuang, V. Redecke, L. She, P. M. Pitha, D. A. Carson, E. Raz, and H. B. Cottam Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: Activation of Toll-like receptor 7 PNAS, May 27, 2003; 100(11): 6646 - 6651. [Abstract] [Full Text] [PDF]
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O. Equils, M. L. Schito, H. Karahashi, Z. Madak, A. Yarali, K. S. Michelsen, A. Sher, and M. Arditi Toll-Like Receptor 2 (TLR2) and TLR9 Signaling Results in HIV-Long Terminal Repeat Trans-Activation and HIV Replication in HIV-1 Transgenic Mouse Spleen Cells: Implications of Simultaneous Activation of TLRs on HIV Replication J. Immunol., May 15, 2003; 170(10): 5159 - 5164. [Abstract] [Full Text] [PDF]
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P.-Y. Bochud, T. R. Hawn, and A. Aderem Cutting Edge: A Toll-Like Receptor 2 Polymorphism That Is Associated with Lepromatous Leprosy Is Unable to Mediate Mycobacterial Signaling J. Immunol., April 1, 2003; 170(7): 3451 - 3454. [Abstract] [Full Text] [PDF]
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J. D. McCurdy, T. J. Olynych, L. H. Maher, and J. S. Marshall Cutting Edge: Distinct Toll-Like Receptor 2 Activators Selectively Induce Different Classes of Mediator Production from Human Mast Cells J. Immunol., February 15, 2003; 170(4): 1625 - 1629. [Abstract] [Full Text] [PDF]
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M. L. Hart, D. A. Mosier, and S. K. Chapes Toll-Like Receptor 4-Positive Macrophages Protect Mice from Pasteurella pneumotropica-Induced Pneumonia Infect. Immun., February 1, 2003; 71(2): 663 - 670. [Abstract] [Full Text] [PDF]
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G. Melmed, L. S. Thomas, N. Lee, S. Y. Tesfay, K. Lukasek, K. S. Michelsen, Y. Zhou, B. Hu, M. Arditi, and M. T. Abreu Human Intestinal Epithelial Cells Are Broadly Unresponsive to Toll-Like Receptor 2-Dependent Bacterial Ligands: Implications for Host-Microbial Interactions in the Gut J. Immunol., February 1, 2003; 170(3): 1406 - 1415. [Abstract] [Full Text] [PDF]
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M. A. Dobrovolskaia, A. E. Medvedev, K. E. Thomas, N. Cuesta, V. Toshchakov, T. Ren, M. J. Cody, S. M. Michalek, N. R. Rice, and S. N. Vogel Induction of In Vitro Reprogramming by Toll-Like Receptor (TLR)2 and TLR4 Agonists in Murine Macrophages: Effects of TLR "Homotolerance" Versus "Heterotolerance" on NF-{kappa}B Signaling Pathway Components J. Immunol., January 1, 2003; 170(1): 508 - 519. [Abstract] [Full Text] [PDF]
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E. K. Brint, K. A. Fitzgerald, P. Smith, A. J. Coyle, J.-C. Gutierrez-Ramos, P. G. Fallon, and L. A. J. O'Neill Characterization of Signaling Pathways Activated by the Interleukin 1 (IL-1) Receptor Homologue T1/ST2. A ROLE FOR JUN N-TERMINAL KINASE IN IL-4 INDUCTION J. Biol. Chem., December 13, 2002; 277(51): 49205 - 49211. [Abstract] [Full Text] [PDF]
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D. Schilling, K. Thomas, K. Nixdorff, S. N. Vogel, and M. J. Fenton Toll-Like Receptor 4 and Toll-IL-1 Receptor Domain-Containing Adapter Protein (TIRAP)/Myeloid Differentiation Protein 88 Adapter-Like (Mal) Contribute to Maximal IL-6 Expression in Macrophages J. Immunol., November 15, 2002; 169(10): 5874 - 5880. [Abstract] [Full Text] [PDF]
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M. R. Horton, S. Boodoo, and J. D. Powell NF-kappa B Activation Mediates the Cross-talk between Extracellular Matrix and Interferon-gamma (IFN-gamma ) Leading to Enhanced Monokine Induced by IFN-gamma (MIG) Expression in Macrophages J. Biol. Chem., November 8, 2002; 277(46): 43757 - 43762. [Abstract] [Full Text] [PDF]
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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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A. D. Edwards, S. P. Manickasingham, R. Sporri, S. S. Diebold, O. Schulz, A. Sher, T. Kaisho, S. Akira, and C. Reis e Sousa Microbial Recognition Via Toll-Like Receptor-Dependent and -Independent Pathways Determines the Cytokine Response of Murine Dendritic Cell Subsets to CD40 Triggering J. Immunol., October 1, 2002; 169(7): 3652 - 3660. [Abstract] [Full Text] [PDF]
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B. Abel, N. Thieblemont, V. J. F. Quesniaux, N. Brown, J. Mpagi, K. Miyake, F. Bihl, and B. Ryffel Toll-Like Receptor 4 Expression Is Required to Control Chronic Mycobacterium tuberculosis Infection in Mice J. Immunol., September 15, 2002; 169(6): 3155 - 3162. [Abstract] [Full Text] [PDF]
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A. Blumenthal, S. Ehlers, M. Ernst, H.-D. Flad, and N. Reiling Control of Mycobacterial Replication in Human Macrophages: Roles of Extracellular Signal-Regulated Kinases 1 and 2 and p38 Mitogen-Activated Protein Kinase Pathways Infect. Immun., September 1, 2002; 70(9): 4961 - 4967. [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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M. Guha and N. Mackman The Phosphatidylinositol 3-Kinase-Akt Pathway Limits Lipopolysaccharide Activation of Signaling Pathways and Expression of Inflammatory Mediators in Human Monocytic Cells J. Biol. Chem., August 23, 2002; 277(35): 32124 - 32132. [Abstract] [Full Text] [PDF]
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T. Wang, W. P. Lafuse, K. Takeda, S. Akira, and B. S. Zwilling Rapid Chromatin Remodeling of Toll-Like Receptor 2 Promoter During Infection of Macrophages with Mycobacterium avium J. Immunol., July 15, 2002; 169(2): 795 - 801. [Abstract] [Full Text] [PDF]
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V. Haehnel, L. Schwarzfischer, M. J. Fenton, and M. Rehli Transcriptional Regulation of the Human Toll-Like Receptor 2 Gene in Monocytes and Macrophages J. Immunol., June 1, 2002; 168(11): 5629 - 5637. [Abstract] [Full Text] [PDF]
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V. S. Carl, K. Brown-Steinke, M. J. H. Nicklin, and M. F. Smith Jr. Toll-like Receptor 2 and 4 (TLR2 and TLR4) Agonists Differentially Regulate Secretory Interleukin-1 Receptor Antagonist Gene Expression in Macrophages J. Biol. Chem., May 10, 2002; 277(20): 17448 - 17456. [Abstract] [Full Text] [PDF]
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B. G. Yipp, G. Andonegui, C. J. Howlett, S. M. Robbins, T. Hartung, M. Ho, and P. Kubes Profound Differences in Leukocyte-Endothelial Cell Responses to Lipopolysaccharide Versus Lipoteichoic Acid J. Immunol., May 1, 2002; 168(9): 4650 - 4658. [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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 B W Jones, K A Heldwein, T K Means, J J Saukkonen, and M J Fenton Differential roles of Toll-like receptors in the elicitation of proinflammatory responses by macrophages Ann Rheum Dis, November 1, 2001; 60(90003): iii6 - 12. [Abstract] [Full Text] [PDF]
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E. Trogan, R. P. Choudhury, H. M. Dansky, J. X. Rong, J. L. Breslow, and E. A. Fisher Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice PNAS, February 19, 2002; 99(4): 2234 - 2239. [Abstract] [Full Text] [PDF]
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