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*
Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Toulouse, France; and
Department of Urology, University of Innsbruck, Innsbruck, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, it was established that phosphoinositol-capped
lipoarabinomannans (PILAMs; Ref. 3) from
Mycobacterium smegmatis and Mycobacterium species
activated murine macrophages in a Toll-like receptor (TLR)-2-dependent
manner (8, 9), leading to the production of
proinflammatory cytokines such as TNF-
(10, 11, 12, 13, 14) and
IL-12 (15). In contrast, mannose-capped lipoarabinomannans
(ManLAMs) isolated from M. tuberculosis and M.
bovis BCG failed to induce a TLR-dependent activation of
macrophages (9) and the production of proinflammatory
cytokines (Refs. 12, 13, 15 ; for review, see Refs.
16, 17, 18). Conversely, M. tuberculosis ManLAMs
were shown to attenuate TNF- and IL-12 expression in human
mononuclear phagocytes (19). Additionally, M.
bovis BCG and M. tuberculosis ManLAMs were found to
bind murine and human macrophages via the mannose receptor (MR; Ref.
20, 21, 22), suggesting a role for ManLAMs in the MR-mediated
adhesion of these mycobacteria to macrophages. However, engagement of
the MR, for example, by mannosylated particulate ligands was not
sufficient to trigger a bactericidal response in macrophages
(23). Furthermore, ManLAMs can effectively induce
expression of the antiinflammatory cytokine TGF-
(13).
All of these findings suggest that ManLAMs may contribute to the
persistence of M. tuberculosis and M. bovis BCG
within macrophages.
In the present study, we assessed whether purified ManLAMs from M. bovis BCG and M. tuberculosis were able to inhibit IL-12 production by human dendritic cells (DCs) stimulated with LPS. The relationship between the structure of ManLAM acyl forms and their capacity to inhibit IL-12 production as well as the role of signals transduced by the MR has been examined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ManLAMs and mannose-capped arabinomannans (ManAMs) from M. bovis BCG Pasteur strain were purified as described previously (24).
Hydrophobic interaction chromatography of ManLAMs
Cellular ManLAMs (cManLAMs; 10 mg) were loaded in 0.1 M ammonium acetate solution containing 15% (v/v) propanol-1 to a octyl-Sepharose CL-4B (Pharmacia, Orsay, France) column (20 x 1.5 cm) preequilibrated with the same buffer. The column was first eluted with 50 ml of equilibration buffer and then with a linear propanol-1 gradient from 15 to 65% (v/v; 250 ml each) in 0.1 M ammonium acetate solution at a flow rate of 5 ml/h. The fractions were collected every 15 min. A total of 20 µl of each fraction was dried and submitted to acidic hydrolysis (100 µl of trifluoroacetic acid 2 M, 2 h at 110°C). The hydrolysates were dried, reconstituted in water, and then analyzed by high pH anion exchange chromatography for arabinose and mannose content.
Preparation of deacylated ManLAMs
Total fraction of ManLAMs (tManLAMs; 200 µg) was incubated in 200 µl of NaOH 0.1 M for 2 h at 37°C. After neutralization with 200 µl of HCl 0.1 M, the reaction products were dialyzed against water.
Preparation of -mannosidase-treated ManLAMs (tManLAMs)
A total of 200 µg of tManLAMs was incubated for 6 h at
37°C in 30 µl of -mannosidase solution (25) (2 mg/ml, 0.1 M
sodium acetate buffer, pH 4.5, 1 mM ZnSO4). After
a second addition of 50 µl of enzyme solution, the reaction was
performed overnight. The reaction products then were dialyzed against
50 mM NH4CO3, pH 7.6.
Elimination of -mannosidase was achieved by denaturation (2 min at
110°C) followed by overnight tryptic digestion (37°C,
trypsin/-mannosidase = 2% by weight). tManLAMs were
recovered after dialysis against water and analyzed for their cap
contents by capillary electrophoresis (CE; see below).
CE and mannooligosaccharide cap quantification
ManLAMs (30–60 pmol) were submitted to mild acidic hydrolysis (15 µl of HCl 0.1 M for 20 min at 110°C; Ref. 24) in the presence of mannoheptose (100 pmol) as internal standard (26). After drying under vacuum, the reaction products were mixed with 0.4 µl of 0.2 M 1-aminopyrene-3,6,8-trisulfonate (APTS; Interchim, Montluçon, France) in 15% acetic acid and 0.4 µl of a 1 M sodium cyanoborohydrure solution dissolved in tetrahydrofuran (27). The reaction was performed for 90 min at 55°C and was quenched by addition of 20 µl of water. From 1 to 5 µl of the APTS derivatives solution was dissolved again in 20 µl of total water before injection in CE. Analyses were performed on a P/ACE CE system (Beckman Instruments, Palo Alto, CA) with the cathode on the injection side and the anode on the detection side (reverse polarity). The electropherograms were acquired and stored on a Dell XPS P60 computer with the System Gold software package (Beckman Instruments).
APTS derivatives were loaded by applying 0.5 psi (3.45 kPa) vacuum for 5 s (6.5 nl injected). Separations were performed with an uncoated fused-silica capillary column (Sigma, Division Supelco, Saint-Quentin-Fallavier, France) with a 50-µm internal diameter and a 40-cm effective length (47 cm total length). Analyses were conducted at a temperature of 25°C with an applied voltage of 20 kV and with acetic acid 1% (w/v), triethylamine 30 mM in water, pH 3.5 as running electrolyte. The detection system consisted of a Beckman laser-induced fluorescence (LIF) equipped with a 4-mW argon-ion laser with the excitation wavelength of 488 nm and emission wavelength filter of 520 nm. The number of each cap motif per ManLAM molecule was determined relative to the internal standard (26).
Nuclear magnetic resonance (NMR) analysis
NMR spectra were recorded on a Bruker AMX-500 spectrometer
(Bruker, Billerica, MA) equipped with an Aspect X32 computer.
Samples were exchanged in
2H2O with intermediate
freeze-drying, then dissolved in
DMSO-2H6 and analyzed in
200 x 5-mm 535-PP NMR tubes at 343 K. The one-dimensional
phosphorus (31P) spectra were measured at 202.46
MHz with phosphoric acid (85%) as the external standard
(
P 0.0). The data were collected in 16,384
k complex data sets, and an exponential transformation (LB
= 2 Hz) was applied prior the processing to 65,536 k real points
in the frequency domain. The ManLAM concentration, scan number (ns),
and spectral width (SW) are indicated in the figure legends.
Fatty acid quantification
ManLAMs (5–6 nmol) were deacylated by strong alkaline hydrolysis (200 µl of NaOH 1 M for 2 h at 110°C) in the presence of pentadecanoic acid (8 nmol) as the internal standard. The reaction was stopped by neutralizing with HCl. Liberated fatty acids were extracted three times with 400 µl of chloroform and after drying under nitrogen stream were methylated with three drops of 10% (w/w) BF3 in methanol (Fluka, Buchs, Switzerland) at 60°C for 5 min. Reaction was stopped by addition of 400 µl of water, and fatty acid methyl esters were extracted three times with 400 µl of chloroform. After drying, fatty acid methyl esters were solubilized in 10 µl of pyridine and trimethylsilylated with 10 µl of hexamethyldisilazane and 5 µl of trimethylchlorosilane at room temperature for 15 min. After drying under nitrogen stream, fatty acid derivatives were solubilized in cyclohexane before analysis by routine gas chromatography (GC) and GC/mass spectrometry (MS) (24). The number of each fatty acid per ManLAM molecule was determined relative to the internal standard.
Generation of monocyte-derived DCs
PBMC were isolated from heparinized whole blood by standard density gradient centrifugation on Ficoll-Paque. Monocytes were isolated from PMBC by centrifugal elutriation (J-6 M centrifuge equipped with a JE-5.0 elutriation rotor; Beckman Instruments). Monocytes (1 x 106 cells) were cultured in 1 ml of RPMI 1640 supplemented with 10% FCS, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM pyruvate, and 5 x 10-5 M 2-ME, as well as 800 IU/ml of each GM-CSF and IL-4. On day 2, 1 ml of fresh medium containing GM-CSF and IL-4 was added. Immature DCs were used on day 5. To induce maturation into CD83+ cells, DCs were cultured with LPS (2 ng/ml) for 48 h.
Measurement of IL-12
Day 5 DCs (3 x 105/ml) were incubated with the stimuli indicated, and IL-12 levels were assessed in DC culture supernatants after 48 h with ELISA specific for either the p40 chain or the p70 heterodimer. IL-12 p70 was measured with matched Ab pairs (Endogen, Woburn, MA). Briefly, anti-p70 IL-12 (3 µg/ml) was plated overnight at 4°C in PBS onto MaxiSorb plates (Nunc, Naperville, IL). Plates were blocked with PBS containing 4% BSA and washed with 0.05% Tween 20 in PBS, pH 7.4. DC culture supernatants were added to the coated wells and incubated for 1 h at room temperature. Wells were washed thoroughly before the addition of a biotinylated anti-IL-12 mAb (500 ng/ml). After 1 h, wells were washed again and then incubated with streptavidin-HRP (1:8000; Zymed Laboratories, San Francisco, CA) for another 60 min. Tetramethylbenzidine (BD PharMingen, San Diego, CA) was used as a substrate, and 2 M H2SO4 was used to stop the enzyme reaction. IL-12 p40 was measured with a commercially available kit (Genzyme, Cambridge, MA). IL-12 was quantitated with a microtiter plate reader.
The capacity to produce cytokines can differ dramatically between individuals (IL-12 p70 production ranged from 0.2 to 5 ng/ml). Therefore, we decided to present the data as percentage of inhibition. The detection limit of the IL-12 p70 ELISA is 15 pg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The total fraction of ManLAMs (tManLAMs) was obtained by
ethanol/water extraction of the disrupted delipidated cells
(16). We first tested the ability of tManLAMs from
M. bovis BCG to stimulate the production of IL-12 by human
monocyte-derived DCs. In addition to abundant MHC molecules, these
cells express the MR (Fig. 1
). In
response to LPS treatment, DCs initiate expression of the CD83 Ag and
up-regulate the T cell costimulator factor (Fig. 1). At 10 and 20
µg/ml, tManLAMs were unable to stimulate the production of the IL-12
p70 heterodimer by DCs (data not shown), consistent with previous
findings in murine macrophages (15).
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). The same concentration of tManLAMs
induced an inhibition of IL-12 p40 production of 
80% (data not
shown).
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ManLAMs present a tripartite structure composed of the GPI anchor,
the polysaccharidic core, and the mannooligosaccharide caps (16, 17). The caps of tManLAMs from M. bovis BCG and
M. tuberculosis are mono-, (1
2)-di-, and
(12)-trimannopyranosides, among which the dimannopyranoside is
the most abundant motif (24, 26, 28). Their presence and
their structure has been evidenced, after mild acidic hydrolysis, APTS
derivatization, and analysis by CE-LIF (24, 26).
To determine whether specific tManLAM domains were involved in their
inhibitory effect of IL-12 production by DCs, degraded tManLAMs were
prepared. tManLAMs were treated with -exomannosidase to obtain
tManLAMs devoid of mannooligosaccharide caps (tManLAMs). The
reaction was controlled by CE-LIF analysis by using the procedure for
cap analysis as described above (24, 26). A typical
electropherogram obtained for -exomannosidase-treated tManLAMs is
presented in Fig. 3a. No
significant peak corresponding to mannooligosaccharide caps was
detected, revealing that tManLAMs are actually devoid of
mannooligosaccharide caps. In contrast to native tManLAMs, tManLAMs
at a dose of 10 µg/ml failed to inhibit IL-12 production by DCs (Fig. 3b). Moreover, PILAMs from M. smegmatis
containing phosphoinositol caps instead of mannooligosaccharide caps
(14), previously described as inducing IL-12 secretion
(15), also were unable to inhibit IL-12 secretion by DCs
(Fig. 3b). Taken together, these results indicated that
mannooligosaccharide caps were critical structural features for the
IL-12 inhibitory effect of ManLAMs.
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), at 10 µg/ml, were unable to inhibit
IL-12 production, indicating that a native acylated GPI anchor was
required for the tManLAM inhibitory effect. In summary, both the
mannooligosaccharide caps and the lipidic moiety of the GPI anchor were
the crucial domains mediating the inhibitory activity of tManLAMs on DC
IL-12 production.
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Fractionation and identification of M. bovis BCG cManLAM acyl forms
cManLAMs were loaded on an octyl-Sepharose column and eluted with
an increasing gradient of propanol-1 in ammonium acetate 0.1 M in
water. The elution profile (Fig. 5b) showed four peaks, two of
weak intensity, peaks A and B, eluting at 27 and 28% propanol-1
respectively, and two of higher intensity, peaks C and D, eluting at
31 and 35% propanol-1 respectively. The fractions corresponding to
the different peaks were pooled, dialyzed, dried, and analyzed by
one-dimensional 31P NMR in DMSO
(31). One-dimensional 31P spectrum
of cManLAMs from peak D (Fig. 5d) exhibited a single
resonance at 1.66 corresponding to phosphate P1 previously
evidenced in the cManLAM mixture (Fig. 5a; Ref.
31). Likewise, one-dimensional 31P
spectrum of ManLAMs from peak C (Fig. 5c) exhibited a single
resonance at 1.83 corresponding to phosphate P3. As previously
established by two-dimensional
1H-31P NMR experiments, P1
and P3 typify ManLAM acyl forms containing at least three (two on the
glycerol and one on O-3 of the myo-inositol) and two fatty
acyl groups (both on the glycerol), respectively (see Fig. 6d, NMR data only).
Unfortunately, the amount of cManLAMs collected in fractions A and B
were not enough to record a one-dimensional 31P
NMR spectrum with significant 31P signal.
However, from the one-dimensional 31P spectrum of
the cManLAMs (Fig. 5a), it can be proposed that cManLAM A
and B correspond to the remaining P5 and P4 phosphates. As noticed
above for P1 and P3, we have previously established that cManLAMs
characterized by P4 and P5 contain at least two and one fatty acids,
respectively (see Fig. 6d, NMR data only). So, from the
elution order of ManLAM A and B, it can be advanced that they
correspond to the at least monoacylated (P5) and diacylated (P4) forms,
respectively.
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A supplementary acylation site, on position 6 of the mannopyranose
(Manp) unit linked to the O-2 of the
myo-inositol, has been reported previously for M.
tuberculosis ManLAMs (32). This data was
established by degradative methods applied on the total ManLAM pool.
Unfortunately, our 31P NMR approach did not allow
us to define the acylation state of this Manp unit (see Fig. 6
d, NMR data only). To tentatively gain access to this
information, the four cManLAM acyl forms were analyzed by
matrix-assisted laser desorption-ionization (MALDI)/MS (not shown). The
mass spectra obtained were similar, all showing a broad peak centered
around 17 kDa with a heterogeneity estimated at 5–6 kDa. This resulted
from the large ManLAM molecular heterogeneity attributable to the
presence of many glyco forms. As a consequence, MALDI/MS analysis was
unsuccessful in determining precisely the presence of a supplementary
acyl group on the purified cManLAM acyl forms. We resorted to a
classical degradative approach. This structural information was
tentatively determined by the assay of the fatty acids liberated by
alkaline hydrolysis and analyzed by GC. As indicated in Fig. 6a, we found 1.6, 2.0, 3.2, and 3.5 fatty acids on cManLAM
A, B, C, and D respectively. So, cManLAM C may contain an acyl group on
the mannosyl unit of the GPI anchor. cManLAMs A and D may also bear
this extra acyl group. As expected, by using this approach, only one
fatty acid was found for the pManLAMs (Fig. 6a). The
presence and the structure of mannooligosaccharide caps on the purified
acyl forms from M. bovis BCG cManLAMs and pManLAMs were
investigated by CE-LIF as noticed above (24, 33, 34). The
number of each mannooligosaccharide cap motif, i.e., mono-,
(12)-di-, and (12)-trimannoside per ManLAM acyl form
was determined relative to an internal standard (Ref. 26 ;
Fig. 6b). All the acyl forms contained identical cap
structures and similar numbers of each cap. Indeed, they shared 1
mannosyl unit, 5–6 dimannosyl units, and 0.5 trimannosyl units (Fig. 6b). It is noteworthy that these caps also were present in
similar amounts in ManAMs (Fig. 6b).
In conclusion, from these structural studies, the five M. bovis BCG ManLAM acyl forms, including pManLAMs and cManLAMs A, B, C, and D, mainly differed by number and the structure of the acyl groups on the GPI anchor.
Effect of ManLAM acylation degree on IL-12 inhibition
The four cManLAM acyl forms were tested for their ability to
inhibit IL-12 production by LPS-stimulated DCs. As shown in Fig. 7, all of the cManLAM acyl forms, at a
concentration of 10 µg/ml, exhibited the same IL-12 inhibitory effect
than tManLAMs. To precisely determine the role of the acyl
groups, dtManLAMs, devoid of IL-12 inhibitory effect (Fig. 4), were
randomly reacylated with palmitic anhydride in pyridine
(35). Obviously, such an acylation is not selective and
occurs on the accessible hydroxyl groups, leading to a large mixture of
acyl forms. The mixture of reacylated ManLAMs showed an inhibitory
effect in the same magnitude as that found for the native tManLAMs (not
shown), suggesting that the fatty acyl residues probably play a key
role in cManLAM clustering.
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ManLAM inhibitory effect on IL-12 release by DCs is mediated by the MR
The data shown in Fig. 3b indicated that
mannooligosaccharide caps were required for ManLAM IL-12 inhibitory
activity, suggesting that the inhibitory signals were transduced by the
MR, which is abundant on DC surface (Fig. 1). To test this hypothesis,
we investigated whether Saccharomyces cerevisiae mannan,
which is known to be a ligand for the MR, was able to inhibit IL-12
production by DCs. At a concentration of 3 mg/ml, S.
cerevisiae mannan completely abolished IL-12 p70 and >50% IL-12
p40 secretion by DCs (Fig. 8a). In addition, to
definitively prove that MR ligation was involved in the inhibition of
IL-12 production, an mAb directed against the MR was tested. Fig. 8b shows that the anti-MR Ab inhibited IL-12 production
by LPS-stimulated DCs in a dose-dependant manner. An isotype-matched
control Ab (IgG1) had absolutely no effect on the LPS-induced IL-12
production (not shown). Because it has been established that
M. bovis BCG and M. tuberculosis ManLAMs bind
murine and human macrophages via the MR (20, 21, 22), these
data strongly indicated that ManLAMs inhibited DC IL-12 production via
binding and cross-linking to the MR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1). Th1 cells
together with cytolytic CD8 T cells are critical to eradicate
intracellular pathogens such as M. tuberculosis (36, 37). Indeed, in humans, IL-12 deficiency leads to a
predisposition to mycobacterial infections despite the formation of
mature granulomas (3). Surprisingly, M.
tuberculosis, the causative agent of human tuberculosis, induces
IL-12 production in vitro from murine phagocytic cells
(38) and human DCs (39) and in vivo at the
human infection site (40). The M. tuberculosis
19-kDa lipoprotein was found to be the potent stimulator of
TLR-2-dependent IL-12 production by human macrophages
(41). However, in vivo, administration of IL-12 to mice
enhances their resistance to M. tuberculosis infection,
suggesting that the level of IL-12 is critical to the control of
M. tuberculosis infection (42, 43).
In the present study, we demonstrate that M. bovis BCG
tManLAMs inhibit IL-12 production in human monocyte-derived DCs,
most likely via MR ligation. This property also was shared by M.
tuberculosis H37Rv tManLAMs (not shown). In contrast, PILAMs from
M. smegmatis, a nonpathogenic mycobacterial strain, did not
inhibit the production of IL-12. ManLAMs and PILAMs share the
same tripartite structure composed of the polysaccharidic core, the GPI
anchor, and the caps (16, 17). To date, the cap structure,
assigned to mannooligosaccharide and phospho-myo-inositol
residues, typify ManLAMs and PILAMs, respectively. Thus,
mannooligosacharide caps appear to play a critical role in the ability
of M. bovis BCG and M. tuberculosis
tManLAMs to inhibit IL-12 production by DCs. This assumption
was further supported by the finding that M. bovis BCG
uncapped tManLAMs, obtained by an -mannosidase treatment, failed to
inhibit IL-12 production. The acyl groups of the GPI anchor appear to
play a crucial role because: 1) M. bovis BCG tManLAM
deacylation abolished IL-12 inhibitory effect; 2) this activity was
restored after reacylation by palmitic anhydride; 3) M.
bovis BCG ManAMs, corresponding to ManLAMs devoid of the GPI
anchor (29), failed to inhibit IL-12 production; and
finally, 4) M. bovis BCG pManLAMs were found to be devoid of
inhibitory activity. It is noteworthy that dtManLAMs as well as
pManLAMs and ManAMs contained the same caps and in similar number as
tManLAMs (Fig. 6b). The abundance of pManLAM acyl form
in the BCG envelope represent only less than 10% of tManLAMs
(24). This is in agreement with the fact that M.
bovis BCG tManLAMs inhibited IL-12 production. M. bovis
BCG pManLAMs (24) differ from M. bovis BCG
cManLAM acyl forms (29) and from M.
tuberculosis H37Rv ManLAM acyl forms (28) by the GPI
anchor bearing a single acyl group tentatively assigned to
12-O-methoxypropanoyl-12-hydroxystearic acid (Ref.
24 ; Fig. 6d). Collectively, these findings
suggested that GPI acyl group structure and GPI acylation degree
modulate ManLAM activity. We previously have established, based on
one-dimensional 31P and two-dimensional
1H-31P NMR experiments,
that M. bovis BCG cManLAMs are composed by a mixture of four
acyl forms, namely A (at least one acyl group, on the glycerol), B (at
least two acyl groups, one on the glycerol and the second one on O-3 of
the myo-inositol unit), C (also at least two acyl groups but
both on glycerol), and D (at least three acyl groups, two on glycerol
and one on O-3 of the myo-inositol unit; Ref.
31 ; Fig. 6d). In this report, the four M.
bovis BCG cManLAM acyl forms A, B, C, and D were successfully
fractionated by hydrophobic interaction chromatography and their
structure characterized by the chemical shift of the
31P resonance. In addition to the three acylation
sites accessible by our 31P NMR experiments,
another potential position has been described in M.
tuberculosis ManLAMs, by Brennan and coworkers (32),
on the O-6 of the mannosyl unit linked on O-2 of the
myo-inositol. Despite the use of nondegradative techniques,
such as NMR or MALDI/MS, our efforts to determine the presence of a
supplementary fatty acid on the purified cManLAM acyl forms were not
successful. As a consequence, this structural information was assessed,
with less accuracy, by fatty acid quantification by GC analysis after
alkaline hydrolysis. Palmitic and tuberculostearic acids were the major
fatty acids liberated, but stearic acid also was recovered in small
amount (Fig. 6a). The determination of the total number of
fatty acids per ManLAM molecule allowed us to propose the final
structures for the cManLAM acyl forms A, B, C, and D as presented in
Fig. 6d. The four purified BCG cManLAM acyl forms A, B, C,
and D, at a concentration of 10 µg/ml, exhibited the same IL-12
inhibitory effect as tManLAMs. The dominant acyl form of H37Rv pManLAMs
and cManLAMs is ManLAM B (28). Likewise, M.
tuberculosis H37Rv pManLAMs and cManLAMs were found to inhibit
IL-12 production. Because the position of the acyl groups seems to be
irrelevant for the inhibitory activity (Fig. 7), it can be concluded
that fatty acid residues are probably involved in the ManLAM
clustering, allowing the mannooligosaccharide caps to be presented
efficiently to DC membrane receptors. This is in agreement with the
finding that the inhibitory effects on IL-12 were mediated by the MR.
Indeed, the involvement of MR in IL-12 inhibition was
supported by 1) the abolition of tManLAM inhibitory effect
after the removing of mannooligosaccharide caps (Fig. 3); 2) the
inhibitory effect of the S. cerevisiae mannan, known to be a
ligand of the MR (Fig. 8a); and 3) the inhibitory effect of
an anti-MR Ab (Fig. 8b). The MR is a type I
transmembrane protein containing eight carbohydrate recognition domains
(CRDs) associated in tandem. It has been established that high-affinity
binding to C-type lectins, such as the MR, requires the cooperation of
the multiple CRDs to bind different terminal mannose residues of the
same multivalent ligand (44). It may be reasonable to
speculate that the differences between M. bovis BCG pManLAMs
and cManLAMs are attributable to differential clustering capacities. An
efficient cManLAM clustering would allow mannooligosaccharide caps from
different cManLAM molecules to bind the MR via multiple CRDs resulting
in high-affinity binding, or more precisely, in high-avidity binding.
In contrast, M. bovis BCG pManLAMs, containing a
single-branched fatty acid, might not cluster, leading to low-affinity
binding to the MR (Fig. 9). Indeed,
pManLAMs and cManLAMs acyl forms have the same mannooligosaccharide
caps and in similar number per molecule, suggesting that the cap
structure was not responsible for the differences in biological
activity. Moreover, differences in binding affinity between M.
bovis BCG pManLAMs and cManLAMs to C-type lectins have recently
been established by Sidobre et al. (25). Indeed, M.
bovis BCG cManLAMs were found to bind to the human surfactant
protein A with a higher affinity than M. bovis BCG pManLAMs
(25). Surfactant protein A is a C-type lectin containing
18 CRD with the same carbohydrate recognition specificity as the MR
(45). From these data, it can be concluded that the ManLAM
capacity to inhibit IL-12 production by DCs is controlled by the
binding affinity to the MR and, consequently, by their capacity to
cluster, i.e., to form large aggregates in water. The capacity of
ManLAMs to form aggregates is evidenced by the line width of their
31P NMR resonance in water. Indeed, in contrast
to M. bovis BCG cManLAMs typified by a large
31P resonance, pManLAMs and dtManLAMs show well
resolved signals indicating that these compounds are poorly aggregated
in water (Ref. 24 ; Fig. 9). Thus, ManLAM clustering seems
to require at least two fatty acyl appendages on the GPI (assumption
corroborated by S. Sidobre, G. Puzo, and M. Rivière, unpublished
results), and the inhibitory effect of cManLAM acyl form A can be
explained by the presence of an extra fatty acid on the GPI mannosyl
unit (Fig. 6, a and d).
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-glucans but not the -mannans are
able to bind CR3, via a cation-independent lectin site
(47), suggesting that CR3 does not mediate ManLAM IL-12
inhibitory effect. The ManLAM effect also could result from a negative
regulation of IL-12 synthesis by the production of antiinflammatory
cytokines such as IL-10 and TGF-. However, PILAMs and ManLAMs
have been shown to elicit TGF- in similar amount from human
monocytes, whereas PILAMs are more potent inducers of IL-10 than
ManLAMs (13).
Therefore, we favor the hypothesis that the inhibition of IL-12
production by DCs in response to ManLAMs is mediated by the MR. The
mechanism by which the MR signaling antagonizes CD14/TLR signaling
remains an interesting question. In all systems where IL-12 suppression
has been molecularly defined, it was found predominantly at the
transcriptional level (7). In fact, IL-12 inhibition
mediated by MR appears to be able to proceed by more than one
mechanism. As noticed above, the signaling pathway of IL-12 stimulation
by LPS and PILAMs (8) requires TLR-4 and TLR-2
respectively. TLRs activate intracellular signaling, most notably via
the transcription factor NF-
B, which results in the induction of
genes coding for effectors of innate immunity such as TNF- and
chemokines (48). The signaling pathway requires the MyD88
adaptor proteins that interact with the cytoplasmic domain of TLR and
recruits IL-1R-associated serine/threonine kinase (49, 50). At present, it is unclear how ManLAMs can interfere with
this signaling pathway through MR ligation. ManLAMs were found to alter
signaling pathway activation of human mononuclear phagocytes by
promoting the tyrosine phosphatase 1 (SHP-1), a tyrosine phosphatase
known to be important for attenuating activation signals
(19). It is conceivable that ManLAMs exert their
inhibitory effects by inducing the dephosphorylation of multiple
proteins, including mitogen-activated protein kinase, involved in the
IL-12 signaling pathway. It also has been reported that ManLAMs induced
nuclear translocation of the transcription factor KBF-1 in murine
macrophages (51). KBF-1 is a homodimer of the NF-B
subunit protein p50 that can function as a transcriptional repressor by
blocking the binding of the NF-B p50-p65 heterodimer to DNA
(52) and consequently the production of IL-12. Finally, MR
also serves as a signaling receptor to elicit transient rise in
intracellular free Ca2+ concentration in macrophages by a
mechanism requiring coligation of both CD14 and MR (53).
Consequently, ManLAM binding to MR and ligation to CD14 could prevent
TLR signaling.
In summary, the present study provides support that ManLAMs are virulence factors that contribute to the persistence of M. bovis BCG and M. tuberculosis in the host and to their maintenance in the human reservoir. This study highlights that the inhibition of IL-12 production by DCs is modulated by the structure of the ManLAM acyl forms. Thus, structural modifications of ManLAM GPI anchor may represent a mechanism for mycobacteria to gain advantage within host tissues.
The activation of macrophages and DCs depends on the net balance between positive and negative signals. Although it is established that the CD14/TLR pathway activates macrophages and DCs, the present study demonstrates that MR ligation negatively regulates DC function and that the MR can transduce inhibitory signals that interfere with TLR-mediated stimulatory signals.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Germain Puzo, Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, 205 route de Narbonne, 31077 Toulouse Cedex 4, France. E-mail address: germain{at}ipbs.fr
3 Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; PILAM, phosphoinositol-capped lipoarabinomannan; TLR, Toll-like receptor; ManLAM, mannose-capped lipoarabinomannan; MR, mannose receptor; DC, dendritic cell; ManAM, mannose-capped arabinomannan; cManLAM, cellular ManLAM; tManLAM, total fraction of ManLAM; tManLAMs, -mannosidase-treated tManLAMs; APTS, 1-aminopyrene-3,6,8-trisulfonate; CE, capillary electrophoresis; LIF, laser-induced fluorescence; NMR, nuclear magnetic resonance; GC, gas chromatography; MS, mass spectrometry; dtManLAM, deacylated tManLAM; pManLAM, parietal ManLAM; Manp, mannopyranose; MALDI, matrix-assisted laser desorption-ionization; CRD, carbohydrate recognition domain.
Received for publication August 31, 2000. Accepted for publication April 13, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) content (b). The 31P
NMR spectra were recorded at 343 K in DMSO-d6. An
exponential multiplication (LB = 2 Hz) was applied before Fourier
transformation. ManLAM concentration was 54 mg/ml (1662 ns;
a), 10 mg/ml (5286 ns; c), 10 mg/ml
(7346 ns; d).
) assays after 48 h. b, DCs
(3 x 105/ml) were incubated with 2 ng/ml LPS and
different amounts of the 19.2 IgG1 anti-MR Ab (BD PharMingen).
Supernatants were harvested for IL-12 p70 assays after 48 h. An
isotype-matched control Ab (IgG1) had absolutely no effect on the
LPS-induced IL-12 production.