1 Lymphocyte development, URA-1961 of the National Center for Scientific
Research, Pasteur Institute, Paris, France
2 Pathogénie Microbienne Moléculaire, Unité INSERM U389,
Institut Pasteur, Paris, France
3 Department of Host Defense, Research Institute for Microbial Diseases, Osaka
University, Osaka, Japan
4 Défense Innée et Inflammation, Unité associée
IP/Inserm Z485, Institut Pasteur, Paris, France
5 Endotoxin Group, UMR-8619 of the National Center for Scientific Research,
University of Paris-Sud, Orsay, France
* Author for correspondence (e-mail: richard.chaby{at}bbmpc.u-psud.fr)
Accepted 8 October 2002
![]() |
Summary |
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Key words: Lipopolysaccharide, Toll-like receptors, Bone marrow, CD14, Innate immunity
![]() |
Introduction |
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It has been established that the activation of monocytes/macrophages by
enterobacterial LPS requires at least four molecules: the serum LPS-binding
protein LBP (Tobias et al.,
1995), the GPI-anchored cell-surface protein CD14
(Wright et al., 1990
;
Golenbock et al., 1993
), the
type I transmembrane Toll-like receptor 4 (TLR4)
(Poltorak et al., 1998
), and
the extracellular molecule MD2 that physically associates with TLR4 on the
cell surface (Shimazu et al.,
1999
). Some investigators have presented this as the paradigm
accounting for cellular responses to LPS. However, other cell types, and
particularly granulocytes, also play a crucial role in host protection.
Furthermore, the considerable diversity of bacteria should incite caution in
granting the status of representative model to a small group of bacteria such
as enterobacteria. Therefore, it is important at the present stage of research
in this area to examine what can occur when other cell types are exposed to
other types of LPSs.
Concerning the responses of other cell types, it has been reported that
Kupffer cells, the resident macrophages of the liver and most abundant tissue
macrophages in the body, do not constitutively express CD14 (Tracy et al.,
1995). They produce TNF- via an LBP-independent and CD14-independent
pathway, when stimulated by nanogram amounts of enterobacterial LPS
(Lichtman et al., 1998
).
Furthermore, when CD14-deficient mice are injected with low doses of LPS,
their liver produces normal levels of acute phase proteins
(Haziot et al., 1998
). In
addition, we reported in previous studies that another mouse cell type, the
bone marrow granulocyte (BMG), is sensitive to very low concentrations of
enterobacterial LPS, although it does not constitutively express CD14
(Girard et al., 1993
).
Exposure of BMGs to this type of LPS actually induces a CD14-independent and
serum-independent stimulation of the cells, and leads to the expression of
CD14 (Pedron et al., 1999
;
Girard et al., 1997
), and
downregulation of L-selectin (Pedron et
al., 2001
) and TNF-receptor 2 (T.P., R.G. and R.C.,
unpublished).
Regarding some LPSs that are structurally and/or functionally different
from those isolated from enterobacteria, it has been shown that they induce a
response in macrophages and B lymphocytes from TLR4-deficient mice (C3H/HeJ
mice), which are unresponsive to enterobacterial LPSs. This is particularly
the case with the LPSs from Pseudomonas aeruginosa
(Pier et al., 1981),
Porphyromonas gingivalis
(Tanamoto et al., 1997
;
Ogawa et al., 1996
) and
Prevotella intermedia (Kirikae et
al., 1999
). In a recent study
(Pedron et al., 2000
), we
found that LPSs from Rhizobiaceae and their structurally atypical
lipid A moieties can stimulate BMGs from TLR4-deficient mice (C3H/HeJ and
C57BL/10 ScCr) and induce the expression of CD14 in these cells.
It was therefore important to determine whether some LPSs, because of
particular structural features of their lipid A moiety, can stimulate cells
via a toll-like receptor distinct from TLR4 or some other receptor unrelated
to the toll family. Recent publications proposed that LPSs from
Porphyromonas gingivalis
(Hirschfeld et al., 2001) and
Leptospira interrogans (Werts et
al., 2001
) may activate macrophages via a TLR2-dependent
mechanism. TLR2 is classically considered as mainly involved in the
recognition of Gram-positive bacteria and mycobacteria
(Yoshimura et al., 1999
;
Flo et al., 2000
). It is also
required in innate host defense to Borrelia burgdorferi, an atypical
Gram-negative bacterium that lacks LPS but abundantly produces lipoproteins
(Wooten et al., 2002
). In
addition to lipoproteins (Hirschfeld et
al., 1999
), porins can also activate immune cells by engaging TLR2
(Massari et al., 2002
).
In the present study, we examined the contributions of TLR4 and TLR2 for
the stimulation of BMGs by structurally atypical LPSs. We focused on LPSs for
which a reliable background of structural data is available. As a follow-up to
our previous studies (Pedron et al.,
2000), we first examined the stimulation induced by the rough-type
LPS of a plant pathogen: Rhizobium species Sin-1. We also examined
LPSs from different strains of Legionella pneumophila, a human
pathogen commonly associated with water-based aerosols and one of the top
three causes of nosocomial pneumonias.
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Materials and Methods |
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Lipopolysaccharides and lipid A
The rough-type LPSs from Salmonella minnesota Re-595 and from
Escherichia coli J5 were from Sigma. The LPS from Bordetella
pertussis (LPS-Bp) and the lipid A fraction of the latter (BpLA), were
prepared as described previously
(Tahri-Jouti et al., 1990).
The LPS from Rhizobium species Sin-1 (`rough' chemotype) and its
lipid A fragment, were provided by R. W. Carlson (University of Georgia,
Athens, GA). The LPS was extracted using hot phenol/water (Westphal and Jan,
1965) and purified by gel-filtration chromatography in the presence of
deoxycholate (Reuhs et al.,
1994
). LPSs from three strains of Legionella pneumophila
serogroup 1 (Philadelphia strain CS338, wild-type OLDA strain RC1 and phase
variant OLDA strain 811) were prepared as described previously
(Lüneberg et al., 1998
;
Zou et al., 1999
;
Kooistra et al., 2002b
). The
lipid A fragments of the Legionella LPSs were prepared by hydrolysis
of 10 mg of LPS in 1 ml of 0.1 M sodium acetate/acetic acid buffer (pH 4.4)
for 4 hours at 100°C. The lipid A fraction was recovered by centrifugation
(2500 g, 15°C, 30 minutes), followed by two washes of the
pellet by resuspension in apyrogenic water and centrifugation as described
above. The washed pellets were lyophilized.
For removal of lipoprotein contaminants, LPSs and lipid A were re-extracted
twice by phenol in the presence of sodium deoxycholate as recommended
(Hirschfeld et al., 2000). The
absence of contaminants in the LPS and lipid A samples was assessed by
SDS-PAGE analysis of the samples and staining the gels with silver nitrate
(Rabilloud et al., 1994
).
Reagents
MALP-2, prepared as described
(Aliprantis et al., 1999), was
from Alexis (San Diego, CA). Tripalmitoyl pentapeptide was from Bachem
(Bubendorf, Switzerland). Rabbit LBP was kindly provided by Richard Ulevitch
(Scripps Research Institute, La Jolla, CA). Mouse recombinant LBP was from
Biometec (Greifswald, Germany). The rat anti-mouse CD14 monoclonal antibody
(Rm-C5-3) was from PharMingen (San Diego, CA). In FACS experiments,
FITC-labeled or biotin-labeled goat anti-rat Ig antibodies (Southern
Biotechnology Associates, Birmingham, AL), and FITC-labeled goat anti-hamster
Ig antibody (Caltag, Burlingame, CA), were used as secondary antibodies, and
biotin-labeled antibodies were stained with FITC-labeled streptavidin
(Amersham-Pharmacia Biotech, Little Chalfont, UK). In western blot
experiments, the biotin-labeled antibody was stained with a
streptavidin-peroxydase conjugate (Southern Biotechnology Associates,
Birmingham, AL). Autoradiography Hyperfilm MP, and all electrophoresis
reagents, including molecular weight standards (rainbow markers), were from
Amersham.
FACS analysis
Bone marrow cells (5x105 cells in 400 µl CM without
FCS) were incubated at 37°C with (10 ng/ml) or without LPS. When used,
inhibitors were added to cell cultures 30 to 60 minutes before LPS. For
detection of membrane antigens, the cells were incubated first (30 minutes,
4°C) with the primary antibody, and stained by reincubation (30 minutes,
4°C) with a labeled secondary antibody. Stained cells were layered on a
50% FCS solution, centrifuged and the cell pellet was resuspended in 0.5 ml of
staining buffer (PBS, 5% FCS and 0.02% sodium azide) containing propidium
iodide (0.2 µg/ml) to stain dead cells. Fluorescent cells were detected by
analysis (5000 cells per sample) on a FACS flow cytometer (FACScan,
Becton-Dickinson Electronic Laboratories, Mountain View, CA) using Cell Quest
Software. Dead cells, which incorporated propidium iodide, were gated out of
analysis. Cells with a fluorescence intensity higher than the maximal level of
auto-fluorescence were scored as fluorescent cells.
Preparation of peritoneal macrophages and TNF- assay
Mice were injected intraperitoneally with 2 ml of 4% thioglycollate (Difco,
Detroit, MI). Three days later, peritoneal exudate cells were isolated from
the peritoneal cavity. Then the cells were cultured for 2 hours and adherent
cells were used as peritoneal macrophages. Peritoneal macrophages
(5x104) were cultured in RPMI-1640 medium supplemented with
10% FCS and exposed to LPS (100 ng/ml) for 24 hours. Concentrations of
TNF- in culture supernatants were determined by ELISA (Genzyme-Techne,
Minneapolis, MN).
Expression vectors
pFLAG-TLR2 and pFLAG-TLR6 were obtained as described previously
(Takeuchi et al., 2001).
Luciferase assay
Human embryonic kidney (HEK) 293 cells were transiently transfected with
the vectors indicated above, together with a pELAM luciferase reporter plasmid
(Takeuchi et al., 2001) and a
pRL-TK (Promega, Madison, WI) for normalization of transfection efficiency by
lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the
cells were stimulated with the LPS from Rhizobium species Sin 1 (100
ng/ml) for 8 hours. Then, the cells were lysed and luciferase activity was
measured using the dual-luciferase reporter assay system (Promega) according
to the manufacturer's instructions.
SDS-PAGE analysis of membrane CD14
Membrane proteins were extracted from the cell pellet with 1% CHAPS in 300
mM NaCl, 50 mM Tris, pH 7.5, supplemented with a cocktail of proteases
inhibitors (aprotinin 10 µg/ml, PMSF 1 mM, pepstatin and leupeptin at 2
µg/ml and iodoacetamide 2 mM.). Solubilized proteins were analyzed by
SDS-PAGE in 10% polyacrylamide slab gels according to the method of Laemmli.
Molecular mass markers from 14.3 to 220 kDa were run in parallel. Gels were
fixed in transfer buffer (20 mM Tris, 150 mM glycine, 20% methanol) and
proteins were transferred onto PVDF membranes (Millipore, Bedford, MA) with a
semidry blotting system at 30 volts for 90 minutes. Membranes were blocked (18
hours at 20°C) with 2% BSA in PBS, and incubated (1 hour, 20°C) with
the rat anti-mouse antibody rmC5-3 (1:1000 in PBS containing 2% BSA). The
blots were washed with 0.1% Tween-20 in PBS, and then incubated for 1 hour at
20°C with a biotin-labeled goat-anti-rat antibody (1:2500 in the same
buffer). After extensive washing and incubation with peroxidase-labeled
streptavidine (1:20,000 in 2% nonfat milk in PBS), sites with peroxidase
activity were detected by chemiluminescence with the Super Signal system
(Pierce, Rockford, IL) according to the guidelines of the manufacturer.
![]() |
Results |
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|
To analyze the effect of the Rhizobium LPS in a more physiological
cellular system than a transfected cell line, we re-examined the response of
BMGs, and particularly the role of TLR2 in this response. We compared the
expression of CD14 in BMGs from normal (C3H/HeOU) and TLR2-deficient
(TLR2-/-) mice. The lipid A fragments of LPSs from Bordetella
pertussis and Rhizobium species Sin-1 were used as inducers
because they represent the biologically active region of the LPSs, and also
because eventual lipoprotein contaminants of LPSs are removed during the
preparation of the lipid A fragments (as observed by SDS-PAGE analysis of a
number of such lipid A preparations). Therefore, BMGs were exposed to lipid A
for 24 hours at 37°C, in the absence of serum. The results of the analysis
of CD14 expression by western blot (Fig.
1B) indicated that, in normal cells, the two lipid A fractions
induced the expression of large amounts of CD14. The B. pertussis
lipid A was also active in TLR2-/- cells. In contrast, a much lower
expression of CD14 was induced by the lipid A from R. species Sin-1
in TLR2-/- cells. This result explains why this atypical LPS was
active in TLR4-deficient mice (Pedron et
al., 2000), and shows that this effect is mediated by TLR2.
LPSs from Legionella induced CD14 expression in BMGs from
normal mice
The structure of the Rhizobium lipid A
(Fig. 2) is atypical. When
compared with the Bordetella pertussis lipid A, which acts via TLR4,
the Rhizobium lipid A exhibits three major differences: the absence
of phosphate groups, a modified sugar backbone (a glucosamine unit is replaced
by 2-aminogluconic acid), and the presence of a very long fatty acid chain
[28:0(27-hydroxy) residue] (Bhat et al.,
1994; Basu et al.,
1999
). In searching for other LPSs with at least one of these
atypical structural features, we chose the Legionella LPSs because
they also contain a long fatty acid chain [27:0(1,27-dioic) or 28:0(27-oxo)
residue]. The three strains of Legionella pneumophila used (CS338,
RC1 and 811) have similar lipid A structures, that differ only in their
heterogeneity in the proportion and length of the different fatty acid chains
(Fig. 2)
(Zähringer et al., 1995
;
Kooistra et al., 2002a
). We
exposed BMGs from C3H/HeOU mice to various concentrations of the three
Legionella LPSs, and analyzed by FACS the expression of CD14. The
results (Fig. 3) show that the
three LPSs from Legionella can induce cell surface expression of
CD14, although the concentrations of these LPSs required for this response (1
µg/ml) were much higher than that at which the B. pertussis LPS
induced a similar effect (0.01 µg/ml).
|
|
Stimulation of BMGs from TLR4-deficient mice by the
Legionella LPSs
To examine the role of TLR4 in the activation of BMGs by
Legionella LPSs, we compared the expression of CD14 by BMGs from
C3H/HeOU, C3H/HeJ and C57BL/10ScCr mice. In a first set of experiments, the
cells were analyzed by FACS after exposure for 24 hours to 1 or 10 µg/ml of
the LPSs. Several experiments gave similar results. As shown in one
representative experiment (Fig.
4A), the three Legionella LPSs induced CD14 expression in
C3H/HeJ and C57BL/10ScCr mice, whereas the B. pertussis LPS did not.
The most efficient activator was the LPS from the L. pneumophila
CS338 strain. In a second set of experiments, expression of CD14 was analyzed
by western blot. The results (Fig.
4B) were consistent with those obtained by FACS, and confirmed
that the three LPSs of Legionella are efficient activators of BMGs
from mice with a TLR4 defect.
|
Influence of the Legionella LPSs on TLR2-deficient
macrophages and BMGs
The capacity of Legionella LPS to activate BMGs from
TLR4-defective mice suggests that another receptor is involved. By analogy
with the the results obtained above with the Rhizobium LPS, we used
TLR2-deficient (TLR2-/-) mice to examine the role of TLR2. We
analyzed first the responses of macrophages. We found
(Table 1) that
thioglycollate-elicited peritoneal macrophages from TLR2-/- mice
did not respond to the TLR2-dependent mycobacterial lipopeptide MALP-2, and
produced nornal levels of TNF- in response to Salmonella and
Bordetella LPSs. In contrast, these cells were not activated by the
Legionella LPSs.
|
The stimulation of BMGs by the Legionella LPSs was then examined.
The expression of CD14 BMGs from TLR2-/- mice was analyzed by FACS,
and compared with that of BMGs from normal mice of the same genetic background
(TLR2+/+). Several experiments gave similar results. As shown in
one representative experiment (Fig.
5A), the LPS isolated from the strain CS338 of L.
pneumophila was completely unable to induce CD14 expression in
TLR2-/- cells. CD14 expression was detectable in TLR2-/-
cells exposed to the two other Legionella LPSs, but the responses
were lower than those of TLR2+/+ cells, and were only induced by
high concentrations of these LPSs (10 µg/ml). These residual responses may
indicate that a receptor distinct from TLR2 reacts with these LPSs or with a
contaminant present in the LPS preparations. To ensure that contaminations
cannot be put forward, the three Legionella LPSs were reextracted
twice by phenol in the presence of sodium deoxycholate
(Hirschfeld et al., 2000).
Because of their hydrophobic nature, the Legionella LPSs are fairly
soluble in phenol, so that only 10% of the LPSs were recovered in the aqueous
phase. However, the ability of these purified (lipoprotein-depleted) samples
to induce CD14 in BMGs of different mouse strains was not different from that
of the non-extracted samples. Analysis of CD14 expression by western blot
(Fig. 5B) confirms that this
cell response to Legionella LPSs is markedly reduced, but not
completely abolished, in TLR2-/- BMGs.
|
Effects of lipid A fragments in TLR4- and TLR2-deficient cells
Some cellular activities of LPS preparations are due to their
polysaccharide region, or are modulated by that region. Other bacterial
components (peptides, lipoproteins) present as contaminants in LPS
preparations can also take part in cell responses
(Hirschfeld et al., 2000).
However, it is generally accepted that LPS effects are those mediated by the
lipid A region of that molecule. To check that this was indeed the case for
the effects of the Legionella LPSs mentioned above, we prepared the
lipid A fragments of these molecules by hydrolysis for 4 hours at 100°C in
a pH 4.4 buffer and washing the insoluble material with water. Analysis of
these lipid A preparations by SDS-PAGE stained with silver nitrate indicated
an almost complete absence of protein contamination (protein content lower
than 0.01%, as determined by comparison of band intensities of protein
standards). BMGs from C3H/HeJ, TLR2+/+, and TLR2-/- mice
were then exposed to these lipid A preparations, and CD14 expression was
analyzed by western blot. The results (Fig.
6) were similar to those induced by the unfragmented LPS
preparations: the three lipids were active in C3H/HeJ cells, and the
activities were markedly reduced in TLR2-/- cells.
|
Because Legionella LPSs are fairly soluble in phenol (see above), whereas their lipid A fraction is not, the lipid A isolated from Legionella pneumophila RC1 was re-extracted twice by phenol in the presence of sodium deoxycholate, to remove residual amounts of unhydrolyzed LPS. This purified lipid A, in which the level of protein contamination was considerably lower than that of a standard E. coli LPS (Fig. 7A), was considerably more active in BMCs from TLR4-defective mice (Fig. 7B). This confirms that pure lipid A from Legionella pneumophila RC1 induces a TLR4-independent effect. We also found that the residual activation of TLR2-/- BMGs induced by the repurified LPS is markedly reduced with the repurified lipid A (Fig. 7C). This observation suggests that the lipid A of Legionella pneumophila RC1 stimulates BMGs exclusively via TLR2, whereas the unfragmented LPS, which contains an additional polysaccharide region, stimulates BMGs via TLR2 and another receptor.
|
Dose-response experiments indicated that even low concentrations (100 ng/ml) of L. pneumophila lipid A can induce detectable levels of CD14 in BMGs from TLR4-deficient (C3H/HeJ) mice. The presence of fetal calf serum (10%), or of rabbit or recombinant mouse LBP (10 µg/ml) did not increase this response (data not shown).
Antagonist effect of Bordetella pertussis lipid A
We established in a previous study
(Pedron et al., 2000) that the
lipid A fragment of the B. pertussis LPS inhibits the expression of
CD14 induced by the lipid A of Rhizobium species Sin-1 in BMGs from
C3H/HeJ mice. As we had previously demonstrated that Rhizobium and
Legionella LPSs are both TLR2-dependent stimulators of BMG, it was
important to determine whether B. pertussis lipid A can also inhibit
the TLR2-dependent response induced by the Legionella LPSs. In a
first experiment, BMGs from C3H/HeJ mice, preincubated for 90 minutes with
various concentrations of B. pertussis lipid A, were exposed for 20
hours to the LPS of L. pneumophila CS338. CD14 expression was
detected by FACS. The results (Fig.
8A) show that 1 µg/ml of lipid A inhibited the stimulation
induced by the LPS of Legionella. In a second experiment, BMGs from
C3H/HeJ mice, preincubated for 2 hours with 10
g/ml of B.
pertussis lipid A, were exposed for 20 hours to the lipid A of L.
pneumophila CS338. CD14 expression was detected by western blot. We found
again (Fig. 8B) that B.
pertussis lipid A induced a partial inhibition of the stimulation
triggered by the Legionella lipid A. Similar results were obtained
with the lipid A fragments of the two other strains of Legionella
(data not shown). In contrast, pre-treatment of the cells with B.
pertussis lipid A (10 µg/ml) did not inhibit CD14 expression induced
in BMGs from C3H/HeJ mice by other TLR2-dependent ligands such as MALP-2 (0.1
to 5 ng/ml) and tripalmitoyl pentapeptide (0.5 to 2.5 µg/ml) (data not
shown). This result suggests that B. pertussis lipid A, which is an
agonist of TLR4, acts also as a specific antagonist of the interaction between
TLR2 and Legionella lipid A.
|
![]() |
Discussion |
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---|
The observation that particular LPSs can activate cells of the immune
system via TLR2 has already been reported by two groups, working on
Porphyromonas gingivalis
(Hirschfeld et al., 2001) and
Leptospira interrogans (Werts et
al., 2001
). The chemical structure of the latter is presently
under investigation, but that of the former has been fully elucidated.
Therefore, comparisons are now possible between the structures of three LPSs
with TLR2-dependent activities: those from Porphyromonas, Legionella
and Rhizobium. Four features can be considered: the number of
phosphate groups, the number of fatty acids, the length of the fatty acids,
and the presence of branched fatty acid chains. The number of phosphate groups
does not seem to be an important parameter for TLR2-dependence since 0, 1 and
2 phosphate groups are present in the lipid A regions of R. species, P.
gingivalis and L. pneumophila, respectively. The number of fatty
acid chains in their lipid A (5, 4 and 6, respectively) does not seem to be
critical either. The length of the fatty acid chains may play some role since
two of these LPSs have one long chain (C28 in R. species
and L. pneumophila) and one LPS has two fatty acids of medium size
(C17 in P. gingivalis). But the most critical feature
seems to be the presence of a substituent or a branch on the penultimate
carbon of a fatty acid chain: a hydroxyl group at C27 in
Rhizobium LPS, a ketone group at C27 in
Legionella LPSs, and a methyl branch at Cn-1 of different
fatty acids in Legionella and Porphyromonas LPSs. These
structural features may also be important for the 3D supramolecular structures
of lipid A (conical, cylindrical or lamellar) (Brandeburg et al., 1993), which
have been postulated to determine differential interactions with TLRs
(Netea et al., 2002
). On the
structural basis mentioned above, we can expect that other TLR4-independent
LPSs such as those of Flavobacterium meningosepticum
(Tanamoto et al., 2001
),
Pseudomonas aeruginosa (Pier et
al., 1981
), and Prevotella intermedia
(Kirikae et al., 1999
) could
also activate cells via TLR2. This would then mean that the presently accepted
paradigm of `LPS activation via TLR4' is no more valid since an increasing
number of examples indicate that structurally different LPSs can activate
cells via different TLRs, and there is no objective reason to consider that
one among the different LPS structures should be privileged, particularly when
we are reminded that Legionella is a frequent cause of human
nosocomial infection.
Our data on the activation of macrophages by Rhizobium LPSs
(Fig. 1A) provide additional
insight into the mechanism of action of this type of LPS. The requirement of
both TLR2 and TLR6 is reminiscent of results obtained with some other
microbial components such as the yeast cell-wall zymosan
(Ozinsky et al., 2000), the
mycoplasmal lipopeptide MALP-2 (Takeuchi
et al., 2001
), the Staphylococcus epidermis modulin
(Hajjar et al., 2001
), and the
Borrelia burgdorferi outer surface protein A lipoprotein
(Bulut et al., 2001
), which all
require both TLR2 and TLR6 for optimal activation of macrophages. This means
that mechanisms triggered by microbial lipopeptides can be extended to some
structurally atypical LPSs, and may suggest that heteromeric associations
between TLR2 and TLR6 can recognize particular LPS structures that are unable
to signal via TLR4.
Another interesting observation of the present study is that Bordetella
pertussis lipid A (BpLA) partially inhibits the activation induced by
Legionella lipid A (LLA) in BMCs from C3H/HeJ mice. One possible
explanation for this observation is that BpLA induces a cross-inhibition via
its interaction with TLR4. Indeed, reduction of surface expression of the
TLR4-MD2 complex has been reported after LPS treatment, even in C3H/HeJ mice
that are hyporesponsive to LPS (Nomura et
al., 2000). Therefore, we can speculate that in BMCs from C3H/HeJ
mice, the downregulation of the TLR4-MD2 complex triggered by BpLA induces a
partial cross-inactivation of TLR2 that lowers the response to the
TLR2-dependent LLA. This would mean that this partial cross-inhibition of TLR2
via TLR4 occurs via a MAL/TIRAP-independent signaling mechanism. Such
inter-TLR cross-talks leading either to synergy
(Beutler et al., 2001
;
Gao et al., 1999
) or to
inhibition/anergy (Sato et al.,
2000
) have already been reported.
However, this hypothesis of cross-inhibition of TLR2 via TLR4 must probably be rejected because we found that BpLA did not inhibit the action of other TLR2-dependent inducers such as MALP-2 and tripalmitoyl pentapeptide. Therefore, another explanation must be proposed for our observation. The most likely is that BpLA behaves as a specific antagonist of LLA on TLR2. This can occur if a partial occupancy of the LPS-binding site of TLR2 by BpLA is not sufficient for triggering the signaling cascade, but makes TLR2 inaccessible for LLA. If this hypothesis is correct, it would mean that TLR2 can interact with many lipid A structures, but only some of these interactions can lead to signaling and cell activation. This hypothesis, and the specificity of the antagonism induced by BpLA, also implies that the TLR2 activator (LLA) and the TLR2 antagonist (BpLA) interact with the same binding site of TLR2, whereas other TLR2 activators (lipopeptides) interact with another binding site on TLR2. Additional investigations are required to confirm this hypothesis.
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Acknowledgments |
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