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B Phosphorylation for Induction of Proinflammatory Cytokines Synthesis by Macrophages Indicates Functional Similarity of Receptors Triggered by Glycosylphosphatidylinositol Anchors from Parasitic Protozoa and Bacterial Lipopolysaccharide1
*
René Rachou Research Center-Fundaçao Oswaldo Cruz and Department of Biochemistry and Immunology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil;
Department of Parasitology, Instituto de Ciências Biológicas, Universidade de São Paulo, São Paulo, Brazil;
Discipline of Cell Biology Biologia Celular, Universidade Federal de São Paulo, São Paulo, Brazil; and
Division of Molecular Parasitology and
¶ Medical Research Council Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12 synthesis by
IFN-
-primed-macrophages exposed to tGPI or tGPI-mucin. In addition,
tGPI-mucin and tGPI were able to induce phosphorylation of I
B, and
the use of SN50 peptide, an inhibitor of NF-B translocation,
resulted in 70% of TNF- synthesis by macrophages exposed to
tGPI-mucin. Finally, the similarity of patterns of MAPK and IB
phosphorylation, the concentration of drugs required to inhibit
cytokine synthesis, as well as cross-tolerization exhibited by
macrophages exposed to tGPI, tGPI-mucin, or bacterial LPS, suggest that
receptors with the same functional properties are triggered by these
different microbial glycoconjugates. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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that are responsible for initiating
IFN- synthesis by NK cells (8, 9, 10). In agreement,
different studies indicate that during the early stages of infection,
before the establishment of acquired protective immunity, the cellular
compartment of the innate immune system plays a crucial role in host
resistance against different intracellular protozoa
(8, 9, 10). To better understand the early stimulation of the innate immune system by parasitic protozoa, studies performed in our laboratories and elsewhere have focused on the identification and chemical characterization of the protozoan products that trigger the proinflammatory and effector functions of macrophages. These studies indicate that tGPI,3 a GPI anchor purified from the mucin-like glycoprotein (tGPI-mucin) of Trypanosoma cruzi trypomastigotes, has an essential role in triggering various macrophage functions (11, 12, 13, 14, 15, 16), similar to the importance of LPS in infection with Gram-negative bacteria. Comparable results were obtained with the GPI anchors purified from Plasmodium falciparum and Trypanosoma brucei (Refs. 17 and 18 ; see review in Ref. 19).
Recent studies have suggested that protozoan GPI anchors may have two
signaling portions (i.e., the glycan core and inositolphospholipid)
that trigger different signaling components responsible for cytokine
and NO synthesis by mammalian host cells (20, 21, 22, 23).
However, not enough information is available regarding the macrophage
receptor(s) and signaling pathways that are triggered by
protozoan-derived GPI anchors. Different studies indicate a similarity
in gene expression and functions displayed by macrophages exposed to
either tGPI anchors, tGPI-mucin, or LPS (11, 12, 13, 14). LPS has
been reported to stimulate signal transduction through the
mitogen-activated protein kinases (MAPKs) (24, 25, 26, 27). The
MAPKs comprise an important group of serine/threonine signaling kinases
that transduce a variety of extracellular stimuli through a cascade of
protein phosphorylations, which lead to the activation of transcription
factors (28, 29, 30, 31). There are at least three distinct MAPK
pathways in mammals, including the extracellular singal-related kinases
(ERK-1/ERK-2), the c-jun N-terminal kinases (JNKs), and the
stress-activated protein kinase (SAPK)-2, also named p38
(30, 31, 32, 33, 34, 35). Here, we compared in a systematic way the
kinetics of phosphorylation of these different members of the MAPK
family, as well as of the inhibitor of the NF-B transcription
factor, IB, in macrophages exposed to tGPI-mucin, tGPI, or LPS
derived from Escherichia coli. In addition, we compared the
ability of drugs that are specific inhibitors of the activation of
ERK-1/ERK-2 and/or SAPK-2/p38, as well as NF-B translocation, to
inhibit the induction of IL-12(p40), TNF-, and NO synthesis by
macrophages exposed to the above-mentioned microbial glycolipids. Our
results show a striking similarity in the macrophage response to LPS
and tGPI-mucin (or tGPI), indicating that the receptors used by these
distinct microbial glycolipids may transduce common signaling
pathways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Five- to 6-wk-old C57BL/6 or C3H/HeJ were obtained from the animal house of Fundaçao Oswaldo Cruz (Rio de Janeiro, Brazil) and maintained under standard conditions in the animal house of the Centro de Pesquisas René Rachou-Fundaçao Oswaldo Cruz (Belo Horizonte, Brazil).
Reagents and Abs
Reagents used were obtained from Sigma (St. Louis, MO) unless
indicated otherwise. SN50 peptide inhibitor of NF-B translocation
and SN50M control peptide; PD 98059, 2'-amino-3'methoxyflavone,
specific inhibitor of ERK-1/ERK-2 phosphorylation; SB 203580,
(4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole,
specific inhibitor of SAPK-2/p38 phosphorylation, were all purchased
from Calbiochem (San Diego, CA). Abs were obtained from the following
sources: anti-IL-12(p40) Abs (clones C17.15 and C15.6, as capture
and detection, respectively) were a generous gift from Dr. Giorgio
Trinchieri (Wistar Institute, Philadelphia, PA); anti-TNF- and
IL-12(p70) kits (Duoset ELISA Development System) were purchased from
Genzyme (Cambridge, MA); and Abs against MAPK family members (i.e.,
ERK-1/ERK-2, SAPK kinase (SKK)-1/MAPK kinase (MKK)-4, and SAPK-2/p38),
I-B, cAMP response element binding protein (CREB)/activating
transcription factor (ATF)-1, and ATF-2 were obtained from New England
Biolabs (Hertfordshire, U.K.).
Purification of T. cruzi-derived tGPI
The tGPI-mucin (GPI-anchored glycoprotein) was isolated from tissue culture trypomastigotes as described previously (16, 36) by using sequential organic extraction followed by hydrophobic-interaction chromatography in octyl-Sepharose column (Pharmacia Biotech, Uppsala, Sweden) and elution with a propan-1-ol gradient (5–60%). The tGPIs were obtained after treatment of tGPI-mucin with proteinase K followed by hydrophobic-interaction chromatography in octyl-Sepharose column and elution with a propan-1-ol gradient (5–60%). Purified tGPI-mucin and tGPI were quantified by myo-inositol analysis (37). The presence of Mycoplasma contaminants in the tGPI-mucin preparations were checked by Edman sequencing and mass spectrometry (matrix-assisted laser desorption ionization-time of flight-mass spectrometry), which did not reveal any contamination with bacterial lipopeptides (16).
Murine macrophage preparation
Thioglycollate-elicited peritoneal macrophages were obtained
from either C3H/HeJ or C57BL/6 by peritoneal washing (11).
Adherent peritoneal macrophages were cultured in 96-well plates (2
x 105 cells/well) at 37°C/5% CO2 in DMEM
(Life Technologies, Paisly, U.K.) supplemented with 10%
heat-inactivated FCS (Life Technologies), 2 mM L-glutamine,
and 40 µg/ml of gentamicin. Cells were incubated with inhibitors of
different MAPK cascade, i.e., PD 98059, an inhibitor of the ERK-1/ERK-2
activation; SB 203580, an inhibitor of the SAPK-2/p38 activation; or
SN50, an inhibitor of the NF-B translocation. The inhibitors were
used on cells at the indicated concentrations for 30 min before
stimulation with LPS (50 ng/ml), tGPI-mucin (10 nM), or tGPI (10 nM)
with or without IFN- (50 IU/ml). Culture supernatants were collected
18 and 48 h after the addition of the microbial product for the
evaluation of TNF- and IL-12(p40)/IL-12(p70) or NO production,
respectively.
Cytokine measurement
IL-12(p40) was determined by ELISA with 5 µg/ml of
anti-IL-12(p40) mAbs: clone C17.15 as the capture Ab and
biotinylated anti-IL-12 (clone C15.6) diluted 750-fold as the
detecting Ab. The development was made with streptavidin-peroxidase
conjugate. The plates were read at 405 nm, and IL-12(p40) concentration
was calculated by reference to a standard curve for murine rIL-12
(11). TNF- and IL-12 were quantified in 18 and 48
h supernatants, respectively, by ELISA with the Genzyme Duoset
kit.
Nitrite measurement
Nitrite concentrations in culture supernatants were assayed at 48 h after macrophage activation by the Griess reaction (38). Plates were read at 550 nm, and NO2 concentration was determined with reference to a standard curve with sodium nitrite in culture medium.
Cell viability assay
To assess toxic effects of the used inhibitors and cell viability we used MTT as described previously (39). Briefly, cells were incubated with 100 µl/well of supplemented medium containing 0.5 mg/ml MTT overnight at 37°C and 5% CO2. Cells then were washed and treated with 100 µl/ml 10% SDS in dimethylformamide:H2O (1:1). Absorbance was read at 570 nm. Cell viability was calculated as relative index of control cells (100% viable cells). No evidence of toxic effects were observed when PD 98059, SB 203580, or SN50 peptide were evaluated in the concentrations used in our experiments.
Lysate preparation
Peritoneal macrophages were cultured and stimulated with either LPS (50 ng/ml), tGPI (10 nM), or tGPI-mucin (10 nM) at the times shown. Where indicated, PD 98059 and/or SB 203580 were added before macrophage stimulation. Cells were washed and lysed on ice in lysis buffer (20 mM Tris-acetate, pH 7.0, 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 10 mM sodium glycerophosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 4 µg/ml leupeptin, 1 mM sodium orthovanadate, 1 mM benzamidine, 0.1% v/v 2-ME, and 2 µM microcystin-LR). Lysates were scraped, collected into Eppendorf tubes, and centrifuged at 13,000 x g for 20 min at 4°C (40).
Electrophoresis and immunoblotting
Cell lysate samples were separated by 10% acrylamide SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked overnight at 4°C with PBS containing 5% (w/v) defatted milk and 0.1% Tween 20. Membranes were washed three times with PBS containing 0.1% Tween 20, then incubated with rabbit polyclonal Abs anti-phosphorylated MAPKs or transcription factors in PBS containing 5% (w/v) BSA and 0.1% Tween 20. After washing, the membranes were incubated with HRP-conjugated anti-rabbit Ab and assayed by the ECL chemiluminescent system (Amersham-Pharmacia Biotech, Little Chalfont, U.K.) according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bacterial LPS is a potent inducer of proinflammatory cytokines by
macrophages, and it recently has been demonstrated that it activates
three different groups of MAPK (ERK-1/ERK-2, JNKs, and SAPK-2/p38) in
cells of the macrophage lineage (24, 25, 26, 27). In our previous
studies, we have shown that the tGPI-mucin or the highly purified tGPI
also activate inflammatory macrophages leading to the production of
proinflammatory cytokines as well as NO (11, 12, 13, 14, 15, 16). Similar
results are obtained with the macrophage cell line, named RAW 264,
resident macrophages, and bone marrow-derived macrophages from mouse
origin (unpublished data). Here, we examined whether the tGPI-mucin or
tGPI were capable of activating these MAPKs by examining their
phosphorylation in immunoblots by using phospho-specific Abs. To be
consistent with our previous publications, we used the inflammatory
macrophages. As shown in Fig. 1
A, ERK-1/ERK-2, SKK-1/MKK-4,
and SAPK-2/p38 phosphorylation were all stimulated by LPS, tGPI-mucin,
or tGPI to a similar extent. The maximum levels of phosphorylation
induced by LPS, as indicated by Western immunoblotting was observed at
30 min for SKK-1/MKK-4 and SAPK-2/p38, whereas the ERK-1/ERK-2 activity
peaked at 15 min poststimulation. The tGPI-mucin strongly activated
ERK-1/ERK-2, which was maximal at 15 min before declining toward the
basal level within 30 min. SKK-1/MKK4 and SAPK-2/p38 were both
stimulated by tGPI-mucin within 15 min and sustained for up to 60 min.
Likewise, tGPI stimulated the three MAPKs. These results clearly show
that either tGPI-mucin or tGPI stimulate all three classes of MAPKs
in murine macrophages.
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We considered that it was important to verify whether the specific
inhibitors PD 98059 and SB 203580 appropriately blocked phosphorylation
of different MAPKs in macrophages activated with tGPI-mucin.
Macrophages were pretreated with a fixed concentration of PD 98059 (40
µM) and/or SB 203580 (10 µM) for 30 min and stimulated with
tGPI-mucin for 15 min. As shown in Fig. 1
B, PD 98059 and SB
203580 inhibited ERK-1/ERK-2 and SAPK-2/p38 phosphorylation,
respectively. PD 98059 (40 µM) significantly inhibited the
tGPI-mucin-stimulated ERK-1/ERK-2 phosphorylation but had no effect on
the phosphorylation of SAPK-2/p38 and SKK-1/MKK-4. In turn, SB 203580
completely abrogated the SAPK-2/p38 activity at 10 µM, and at the
same time appeared to increase the levels of ERK-1/ERK-2 and
SKK-1/MKK-4 phosphorylation.
Effect of drugs inhibiting phosphorylation of ERK-1/ERK-2
and SAPK-2/p38 on the production of IL-12, TNF-, and NO
To discriminate between the roles of ERK-1/ERK-2 and SAPK-2/p38 on
the production of cytokines and NO after stimulation with tGPI-mucin
and tGPI, we investigated the effects of PD 98059 (34), a
specific inhibitor of the MAPK cascade that leads to ERK-1/ERK-2
phosphorylation, and SB 203580 (35), an inhibitor of
SAPK-2/p38 phosphorylation and activity. Monolayers of macrophages were
pretreated with PD 98059 or SB 203580 30 min before the addition of
LPS, tGPI-mucin, and tGPI. As shown in Fig. 2A, neither PD 98059 nor SB
203580 inhibited NO production in response to stimulation by LPS,
tGPI-mucin, or tGPI. This is in agreement with a previous study showing
that the NO production induced by TNF- or LPS in murine macrophages
was not affected by PD 98059 or SB 203580 pretreatment (41, 42). In contrast, the LPS-induced TNF- production was
inhibited in 30% by PD 98059 at 40 µM. The TNF- inhibition
observed in tGPI-mucin- or tGPI-treated macrophages was lower, reaching
25% at the highest dose of PD 98059. Preincubation with SB 203580
of LPS-, tGPI-, or tGPI-mucin-treated macrophages resulted in a
significant inhibition of TNF- production in a dose-dependent
manner. The maximum inhibitory effect was 60% at 10 µM.
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A show enhanced IL-12(p40) production, up
to 40% in the PD 98059-treated cells. In contrast, SB 203580 had a
small inhibitory effect on IL-12(p40) production by the stimulated
macrophages in a dose-dependent manner with IC50
>10 µM. Similar results were obtained for the synthesis of
IL-12(p70) (data not shown).
In other experiments, IFN--primed or unprimed macrophages were
treated with a combination of PD 98059 (40 µM) and SB 203580 (10
µM) followed by LPS, tGPI, or tGPI-mucin stimulation and monitoring
nitrite and cytokine production (Fig. 2B). Although there
was no inhibition of NO synthesis with both inhibitors, the combination
of PD 98059 and SB 203580 resulted in 85% inhibition of TNF-
production. As mentioned above, PD 98059 appeared to increase IL-12
production by primed macrophages after LPS, tGPI, or tGPI-mucin
stimulation but enhanced SB 203580-mediated inhibition of IL-12
production.
Activation of CREB and ATF-2 in macrophages stimulated with tGPI-mucin
The activation of the MAPK pathways results in changes in gene
expression mediated by activating various transcription factors. So we
investigated the activation of CREB, ATF-1, and ATF-2, which can be
activated by SAPK/JNK and SAPK-2/p38 in response to inflammatory
cytokines and stress stimuli. As shown in Fig. 3A, tGPI-mucin induced the
phosphorylation of CREB and ATF-2. The phosphorylation of CREB and
ATF-2 peaked at 30 min, and the signal was sustained up to 60 min after
macrophage stimulation. The Ab we use to detect activation of CREB also
recognizes phosphorylated ATF-1. However, in the conditions in which
our experiment were performed, we were unable to detect ATF-1
activation in macrophages exposed to tGPI-mucin.
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B). Consistent with the results shown on cytokine synthesis
(Fig. 2), SB 203580 presented a greater inhibitory effect than PD 98059
on CREB phosphorylation. More important, SB 203580 and PD 98059 had an
additive inhibitory effect on tGPI-mucin induced CREB phosphorylation.
By contrast, the phosphorylation of ATF2 was unaffected by the use of
PD 98059 or SB 203580.
Involvement of NF-B in cytokine production by macrophages in
response to tGPI-mucin
We have also investigated whether the tGPI-mucin is capable of
activating the NF-B transcription factor. For this purpose,
peritoneal macrophages were stimulated by LPS, tGPI, or tGPI-mucin for
different intervals of time, and NF-B release was indirectly
evaluated through IB phosphorylation. As shown in Fig. 4A, IB phosphorylation
occurred rapidly after LPS, tGPI (not shown), or tGPI-mucin
stimulation. To examine the involvement of NF-B in tGPI-mucin- or
LPS-induced cytokine and NO secretion by macrophages, we used SN50, a
cell-permeable peptide, which inhibits the NF-B translocation to the
cell nucleus. The NF-B nuclear translocation is maximally inhibited
at 18 µM (44). As depicted in Fig. 4B, SN50
inhibited 70% of the TNF- production at 18 µM. To verify the
specificity of the effect observed, we used SN50M, the peptide control,
which did not affect the TNF- production. By contrast, SN50 appeared
to have a marginal effect on the IL-12(p40) synthesis with a maximal
inhibition at 18 µM. Preincubation of the cells with SN50 had no
effect on the release of NO after stimulation with LPS, tGPI (not
shown), or tGPI-mucin. These data suggest that NF-B is a main
transcription factor involved in induction of TNF-, but not
IL-12(p40) or inducible NO synthase (iNOS) transcription.
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Previous studies have revealed that pretreatment of macrophage in
vitro with LPS also induce a refractory state subsequent to stimulation
with LPS, which includes the inhibition of MAPKs (i.e., ERK-1/ERK-2,
JNKs, and SAPK-2/p38) and IB phosphorylation (45). This
study also showed cross-tolerance between IL-1
and LPS that use
functionally similar receptors. Therefore, we decided to perform
desensibilization experiments to investigate whether tGPI-mucin and LPS
exhibit cross- tolerization. Macrophages from C57BL/6 mice were
pretreated with LPS or GPI-mucin for 20 h, restimulated with
either LPS or tGPI-mucin, and TNF-, IL-12(p70), and NO synthesis
evaluated thereafter. The results presented in Fig. 5 demonstrate that pretreatment with LPS
or GPI-mucin resulted in partial and complete inhibition of TNF- and
IL-12 in response to the second stimulation, independent of the nature
of the microbial stimuli. Collectively, these results further suggest
that LPS and tGPI-mucin use functionally similar receptor to induce
both TNF- and IL-12 synthesis by inflammatory macrophages. In
contrast, no desensibilization was observed in terms of NO
production.
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B in C3H/HeJ-derived macrophages
exposed to tGPI
LPS was shown to mediate cellular activation by a member of the
human Toll-like receptor (TLR) family (46). TLR4 and TLR2
have been implicated in the response of cells to LPS and other
bacterial glycolipids/lipopeptides, respectively (47, 48).
Because the pattern of macrophage activation by the protozoan-derived
GPI is analogous to that by LPS, we speculated that signal transduction
by TLR might also be triggered by GPI anchor binding. The TLR4 has been
shown to be mutated in C3H/HeJ mice, which are low responders to LPS
(49). Therefore, we assessed the tGPI-mucin ability to
induce MAPK activation in macrophages from C3H/HeJ mice. As shown in
our previous studies (11, 12, 16) and in Fig. 6A, high levels of TNF-,
IL-12, and NO are produced by macrophages from C3H/HeJ mice exposed
to tGPI-mucin or tGPI, but not to LPS. C3H/HeJ macrophages
then were treated with LPS or tGPI-mucin for 15 or 30 min, and cell
lysates were tested for ERK-1/ERK-2, SKK-1/MKK-4,
SAPK-2/p38, and IB activation by measuring their respective
phosphorylation. As expected, LPS did not induce any MAPK or IB
phosphorylation in these cells. However, the absence of functional TLR4
receptor did not affect the tGPI-mucin-induced MAPK and IB
phosphorylation (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To use a more defined system to investigate the signaling pathways
involved in cytokine synthesis by macrophage exposed to T.
cruzi, we tested the ability of tGPI-mucin or highly purified tGPI
to trigger phosphorylation of different MAPKs, IB, and the
involvement of these pathways on cytokine synthesis. In the present
study, we demonstrated that tGPI-mucin or tGPI are capable of
triggering phosphorylation of ERK-1/ERK-2, SKK-1/MKK-4, and SAPK-2/p38,
as well as IB in mouse peritoneal macrophages. As tGPI-mucin (or
tGPI) induced the same pattern of cytokine release as LPS in murine
macrophages, we compared the effect of LPS or tGPI-mucin/tGPI on the
kinetics of MAPK and IB phosphorylation in these cells. The
phosphorylation of different MAPKs was similar when murine macrophages
were exposed to distinct microbial glycolipids. By using specific
inhibitors, we investigated the contribution of ERK-1/ERK-2 and
SAPK-2/p38 in the cytokine and NO synthesis induced in macrophages
stimulated by bacterial or protozoan glycolipids. Taken together, our
results suggest that SAPK-2/p38 and to a lesser extent the ERK-1/ERK-2
pathways are involved in the synthesis of TNF- by stimulated
macrophages. These conclusions are in agreement with published data in
another system (50, 51, 52, 53). Simultaneous inhibition of
ERK-1/ERK-2 and SAPK-2/p38 resulted in 75% inhibition of TNF-
release by macrophages exposed to tGPI-mucin or tGPI. Our experiments
with SB 203580 and PD 98059 also support the findings that CREB is a
main physiological substrate of ERK-1/ERK-2 and SAPK/p38
(54) and the hypothesis that this transcription factor may
be an important element controlling TNF- synthesis by macrophages
exposed to microbial glycolipids (55).
In our experiments, IL-12 production was only slightly sensitive to the SAPK-2/p38 inhibitor, suggesting a minor positive regulatory role of this MAPK on IL-12 synthesis stimulated by microbial glycolipids. In contrast, Lu et al. (56) have shown a defective production of the IL-12 in mitogen-activated MKK3 (specific upstream MAPK for SAPK-2/p38)-deficient mice. Thus, it is possible that MKK3 may also activate an unknown SB-insensitive pathway, which is also responsible for induction of IL-12 synthesis. In contrast, we found a stimulatory effect of PD 98059 on IL-12 production by macrophages exposed to tGPI-mucin, tGPI, or LPS, suggesting that the IL-12 synthesis is negatively regulated by the ERK-1/ERK-2 pathway. In fact, Feng et al. (43) have suggested that Leishmania may suppress resistance to infection by switching on the ERK-1/ERK-2-mediated negative regulation of IL-12 production, hence preventing generation of a protective Th1 immune response.
The specific role of different MAPKs on iNOS induction and NO
production have produced contrasting results. Da Silva et al. have
shown that SAPK-2/p38 is necessary but not sufficient for iNOS
induction by TNF- and IL-1- stimulation (57).
ERK-1/ERK-2 were shown to be necessary in the iNOS regulation by
IL-1 and IFN- (58), but had no effect on LPS/IFN-
induction of the enzyme (59). In glial cells, the
induction of iNOS expression and NO synthesis by IFN- and LPS was
partially blocked by inhibiting ERK-1/ERK-2 and SAPK-2/p38 with PD98059
or SB203580, respectively, and almost completely blocked in the
presence of both inhibitors (60). However, in the present
paper, no effect on NO production was observed by using specific
antagonistic drugs of ERK-1/ERK-2 or SAPK-2/p38 phosphorylation with
macrophages costimulated either with tGPI, tGPI-mucin, or LPS and
IFN-. Our findings are in agreement with previous studies showing
that the iNOS induction by LPS in macrophages is unaffected by PD98059
and/or SB203580 (41, 42).Thus, the protein kinase that is
rate-limiting for iNOS transcription appear to vary from cell to cell
and/or according to the stimuli used in the different studies.
The crucial role of NF-B in cytokine induction was established by
using the peptide SN50, which inhibits the translocation of NF-B. An
inhibition of 70% of the TNF- production was observed when
tGPI-mucin or LPS-stimulated macrophages were pretreated with 18 µM
of SN50. These findings are in agreement with early studies showing
that NF-B is an important transcription factor required for maximal
induction of TNF- synthesis (61). In contrast, we found
that SN50 has only minor or no effect on induction of NO or IL-12(p40)
synthesis by IFN--primed macrophages exposed to the different
microbial stimuli. A half site for NF-B has been identified and
described in the IL-12(p40) promoter (62); however, the
role of NF-B on induction of IL-12 is poorly understood. Consistent
with our findings, Feng et al. (43) concluded that NF-B
binding may not be necessary or sufficient for induction of iNOs but
rather reinforces the idea that IFN regulatory factor complex may be
the major regulatory factor. Altogether, the results presented here
suggest that in our system, NF-B plays a major role in induction of
TNF-, but not IL-12 or iNOS expression.
The recognition system for the stimulatory GPI-mucin appears to share
much in common with the recognition system for LPS. LPS and tGPI or
tGPI-mucin trigger the same pattern of phosphorylation of different
members of the MAPK family. In addition, similar
IC50 values of inhibitors specific for different
MAPKs and NF-B were necessary to inhibit different functions (i.e.,
cytokine) in macrophages exposed to either LPS, tGPI-mucin, or tGPI.
Furthermore, our study demonstrates that pretreatment of mouse
macrophages with either LPS or tGPI-mucin effectively induced a
state of cross-tolerance as evidenced by significantly lower TNF-
and IL-12 release in response to each one of these stimuli. The finding
of cross tolerance may also indicate the similarity of the
receptors triggered by LPS and tGPI-mucin in inflammatory macrophages
(45).
Studies have demonstrated the importance of members of the TLR family
in the macrophage response to bacterial glycolipids
(46, 47, 48, 49). The role for TLR4 in LPS-induced activation in
macrophages is supported by the demonstration that a mutation in the
gene for TLR4 is associated with LPS hyporesponsiveness in the C3H/HeJ
(49). Indeed, the results presented here with macrophages
from C3H/HeJ mice demonstrate that LPS-induced phosphorylation of
ERK-1/ERK-2, SKK-1/MKK-4, and SAPK-2/p38 is dependent on functional
TLR-4. Interestingly, phosphorylation of MAPKs and IB was still
observed in macrophages from C3H/HeJ mice exposed to tGPI-mucin or
tGPI. Thus, our results indicate that although functionally similar,
the receptors triggered by LPS and tGPI are different. Considering that
various members of TLR have been cloned at the moment, we speculate
that T. cruzi-derived GPI anchors may in fact engage a
distinct member of the TLR family, the nature of which is being
investigated in our laboratories.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ricardo T. Gazzinelli, Laboratory of Immunopathology, Centro de Pesquisas René Rachou, Fundaçao Oswaldo Crus, Av. Augusto de Lima 1715, Barro Preto, 30190-002, Belo Horizonte, MG, Brazil.
3 Abbreviations used in this paper: tGPI, GPI anchor purified from tGPI-mucin; tGPI-mucin, GPI-anchored mucin-like glycoproteins derived from Trypanosoma cruzi trypomastigotes; CREB, cAMP response element binding protein; ATF, activating transcription factor; ERK, extracellular signal-related kinase; MAPK, mitogen-activated protein kinase; JNK, c-jun N-terminal kinase; iNOS, inducible NO synthase; SAPK, stress-activated protein kinase; SKK, SAPK kinase; MKK, MAPK kinase; TLR, Toll-like receptor.
Received for publication June 13, 2000. Accepted for publication December 21, 2000.
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References |
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), tGPI (10 nM;
), or LPS (50 ng/ml; •). Levels of TNF-
) or not (
