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*
Department of Immunology, University of Glasgow, Glasgow, United Kingdom; and
Department of Chemistry, University of Dundee, Dundee, United Kingdom
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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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NO, which mediates many of the nonspecific cytotoxic and inflammatory
responses of macrophages following infection by pathogens, is generated
following the up-regulation of expression of the inducible form of NO
synthase (iNOS).3
Similarly, the biological activity of IL-12 (a heterodimeric cytokine
comprising p35 and p40 subunits), which is the key cytokine driving Th1
cell development (3, 10), is regulated by the induction of
the p40 subunit (11, 12). Expression of these
immunomodulatory proteins appears to be regulated primarily at the
level of transcription (13, 14, 15, 16, 17, 18), and, indeed, NF-
B
activation has been shown to be a key factor in the regulation of
induction in both cases (12, 19). Similarly, IFN
regulatory factors (IRFs) also appear to play a key role in the
induction of both of these macrophage effectors (Refs. 15 ,
20 , and 21 and our unpublished data) via the
IFN-stimulating response element in iNOS (13) and the
nuclear complex termed F1, which includes multiple nuclear factors
(IRF-1, c-Rel, and GLp109) and acts at the Ets site in the IL-12
promoter (17). However, little is known about the early
signaling events underlying the up-regulation of iNOS or IL-12 (p40)
expression by LPS in macrophages.
LPS has been reported to stimulate a variety of signal transduction elements such as the Src-related protein tyrosine kinases (22, 23), phospholipases (24, 25), protein kinase C (26, 27), raf (28, 29), and mitogen-activated protein (MAP) kinases (30, 31, 32, 33) in a variety of cellular systems. Although many of these signals have not been consistently reproduced in macrophages (32), it is clear that LPS can stimulate MAP kinases in these cells (27). The MAP kinases are an important group of serine/threonine signaling kinases that, by modulating the phosphorylation, and hence activation status of transcription factors, link transmembrane signaling with gene induction events in the nucleus (34, 35). In mammalian cells, three major subgroups of MAP kinases have been identified, and these comprise the extracellular signal-regulated kinases (ERKs), the c-jun amino-terminal kinases (JNKs), and the p38 MAP kinases (36). These MAP kinases are all activated by phosphorylation of a common threonine-X-tyrosine regulatory motif by their distinct upstream dual-specificity (Thr/Tyr) MAP kinase kinases (37, 38, 39). Although LPS has previously been reported to activate all three types of MAP kinases, the physiological relevance of such MAP kinase signaling to macrophage function remains unclear.
In this study, we show that LPS stimulates, with differential kinetics, the activation of ERK, JNK, and p38 MAP kinases in J774 macrophages. Moreover, we demonstrate, by the use of cell-permeable inhibitors selective for the ERK (PD98059) (40) and p38 (SB203580) (41) signaling cascades, that such ERK and p38 MAP kinase activation plays differential roles in the regulation of LPS-stimulated induction of IL-12 (p40) and iNOS gene expression in macrophages. The physiological relevance of such MAP kinase regulation of macrophage effector function is demonstrated by our finding that synthetic Leishmania lipophosphoglycan exerts its inhibitory effects on the production of IL-12 by macrophages through stimulation of ERK MAP kinase, which acts to suppress transcription of IL-12 (p40).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents used were obtained from Sigma (Poole, U.K.) unless
indicated otherwise. PD98059 and SB203580 were obtained from Alexis
(Nottingham, U.K.). Abs used were obtained from the following sources:
monoclonal anti-maciNOS (Transduction Laboratories, Affiniti,
Exeter, U.K.); monoclonal anti-IL-12 (p40) (Genzyme, Cambridge,
U.K.); polyclonal anti-IL-12 (p70) Ab (raised in our laboratory
using rIL-12 (p70)). Abs against all three MAP kinase family members
were obtained from New England Biolabs (Hertfordshire, U.K.). Abs
against IRF family proteins, NF-B family proteins, and Ets1/2 were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
HRP-conjugated anti-mouse IgG and anti-rabbit IgG abs were
obtained from the Scottish Ab Production Unit (Lanarkshire, U.K.).
Purification of murine peritoneal macrophages, cell culture, and cytokine measurement
Thioglycollate-elicited peritoneal macrophages were removed from
BALB/c or 129 mice by peritoneal washing and enriched by plastic
adherence. Adherent peritoneal macrophages or the murine macrophage
cell line, J774 (American Type Culture Collection, Manassas, VA), were
cultured at 37°C/5% CO2 in DMEM (Life
Technologies, Paisley, U.K.) supplemented with 10% heat-inactivated
FCS (Life Technologies), 2 mM L-glutamine, 50 U/ml of
penicillin, and 50 µg/ml of streptomycin. Cells were cultured in
96-well plates and incubated with or without the MEK inhibitor,
PD98059, or the p38 inhibitor, SB203580, at the indicated
concentrations for 1 h before stimulation with LPS or LPS plus
IFN-
. Culture supernatants were collected 24 h after LPS
stimulation, and IL-12 (p40) was assayed by ELISA using paired Abs
(Genzyme). IL-12 (p70) was measured using an ELISA kit from PharMingen
(San Diego, CA).
Synthetic phosphoglycan (sPG)
Synthetic fragments
([Gal(ß1–4)Man(
)-PO3H.NH3]10-OH.
NH3) of Leishmania lipophosphoglycans,
which have previously been shown to mimic the actions of the natural
parasite molecules (8, 9), were synthesised as described
previously (42).
NO2 measurement
Nitrite concentrations in culture supernatants were determined by Griess reaction as described previously (43).
Preparation of cell lysates
Stimulated cells (1–2 x 107/sample) were washed twice with ice-cold TBS (25 mM Tris.Cl, pH 7.4, 150 mM NaCl, and 100 mM sodium orthovanadate) and harvested with a plastic scraper. The cells were lysed in lysis buffer (25 mM Tris buffer, pH 7.4, containing 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM EDTA, 2 mM EGTA, 10 mM NaF, 1 mM DTT, 50 µg/ml each of leupeptin, aprotinin, and PMSF) by incubation on ice for 30 min. Lysates were then centrifuged at 13,000 x g at 4°C for 10 min, and the supernatants were transferred to fresh tubes and stored at -70°C until required. Protein concentrations of the lysates were determined using the Coomassie protein assay reagent (Pierce, Rockford, IL).
Western blot analysis
Cell lysates were resolved by SDS-PAGE before transfer to nitrocellulose using a transblot system (Hoffer Scientific SE600, San Francisco, CA). Nitrocellulose filters were then incubated with washing buffer (0.01 M Tris-HCl buffer, pH 7.5, containing 0.1 M NaCl and 0.1% Tween 20) containing 2% BSA for at least 1 h to block nonspecific protein binding. Primary Abs were diluted in washing buffer containing 1% BSA and applied to the filter for 1 h at room temperature or overnight at 4°C. Following washing, the blots were incubated with the appropriate HRP-conjugated secondary Ab (diluted up to 1:5000 in wash buffer containing 1% BSA) for 1 h at room temperature. Immunoreactive bands were visualized by the enhanced chemiluminescence system (Amersham, Buckinghamshire, U.K.).
Northern blot analysis
IL-12 (p40) and ß-actin cDNA fragments amplified from J774
cells by RT-PCR were cloned into TA vector (Novagen, Abingdon, U.K.),
confirmed by DNA sequencing. These cDNA probes were labeled with
[-32P]dATP using a commercial random priming
kit (Life Technologies). Macrophage total cellular RNA was purified
using the Bio/RNA-Xcell method (Biogenesis, Cambridge, U.K.), and the
concentration of RNA in samples was estimated by UV spectroscopy.
Northern hybridization analysis was conducted using radiolabeled IL-12
(p40) cDNAs as specific probes, and the ß-actin probe was used for
reblotting as a loading control.
EMSA
Nuclear extracts were prepared essentially as described
previously (44, 45, 46). Briefly, treated cells were washed
twice with ice-cold TBS before resuspending in 400 µl cold buffer A
(10 mM HEPES buffer, pH 7.9, containing 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1 mM DTT, 0.5 mM PMSF, 100 µM sodium orthovanadate). Cells were
allowed to swell on ice for 15 min, after which 25 µl of a 10%
solution of Nonidet P-40 (Fluka, Poole, U.K.) was added and the cells
were vortexed vigorously for 15 s. The homogenate was centrifuged
at 13,000 x g for 30 s, and the resulting nuclear
pellet was resuspended in 100 µl ice-cold buffer B (20 mM HEPES
buffer, pH 7.9, containing 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT,
1 mM PMSF, and 100 µM sodium orthovanadate) and rocked vigorously at
4°C for 15 min. The nuclear extract was centrifuged for 5 min at
13,000 x g at 4°C, and the supernatant was collected
and frozen in aliquots at -70°C until use. Binding reactions were
initiated by preincubation of nuclear extract protein (10 µg) with
double-stranded poly(dI-dC) (200 ng, Pharmacia Biotech, St. Albans,
U.K.) in a reaction buffer (20 mM HEPES buffer, pH 7.9, containing 40
mM KCl, 1 mM MgCl, 0.1 mM EGTA, 0.5 mM DTT, 10% glycerol, and 0.1%
Nonidet P-40) for 20 min on ice in a volume of 30 µl. The appropriate
radiolabeled synthetic oligonucleotide DNA probe (0.5 ng in 1 µl) was
then added, and the mixture was incubated on ice for a further 20 min.
For Ab supershift assays, Ab (1 µl) was added during the
preincubation period. The resultant DNA-protein complexes were resolved
by nondenaturing 6% PAGE and the gels dried and visualized by
autoradiography. The oligonucleotide probes used in this work are:
probe D (5'-CCCAACTGGGGACTCTCCCTTTGG-3'), NF-B site on murine
iNOS promoter region; probe M (5'-CAAAACATTCTGGGGGAATTTTAAGA-3'),
NF-B site on murine IL-12 (p40) promoter region; probe G
(5'-CACTGTCAATATTTCACTTTCATAAT-3'), an IRF-E in iNOS promoter;
probe K (5'-CAGAGATACTAATTTCTGTTTACATCATGCCTAAGGT-3'), a sequence
that binds to F1 in the murine IL-12(p40) promoter.
Statistical analysis
Statistical significance (p < 0.05 or p < 0.01) of differences between treatment groups was assessed by the Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate whether the ERK, JNK, and p38 MAP kinase pathways
are involved in LPS signal transduction in macrophages, we examined the
activation of the three MAP kinases by detecting their dually
phosphorylated (Tyr/Thr) forms by Western blotting using specific
anti-phosphokinase abs. LPS strongly stimulated a rapid (within 1
min) and transient increase in the levels of activation of both ERK-1
(3.7-fold) and ERK-2 (3.2-fold) MAP kinase activities, which peaked at
10 min but remained elevated for at least 60 min above the low basal
levels of ERK activation observed in unstimulated J774 cells (Fig. 1
, A and B).
Although there was no basal activity of ERK MAP kinases in murine
peritoneal macrophages, LPS strongly stimulated both ERK-1 and ERK-2
activation within 10 min. In contrast, IFN- only weakly stimulated
ERK activity and appeared to suppress LPS-stimulated ERK
phosphorylation (Fig. 1C). Little or no activation of p38
MAP kinase could be detected in unstimulated J774 cells. LPS strongly
activated p38, which was maximal at 10 min and sustained for up to 60
min (Fig. 1, D and E). Likewise, p38 was
activated following stimulation of murine peritoneal macrophages with
either LPS or LPS plus IFN- for 10 min (Fig. 1F).
Similarly, although stress-activated protein kinase (SAPK)/JNK MAP
kinases were found not to be significantly activated in either
unstimulated J774 cells or murine macrophages, LPS, following a lag
period of up to 10 min, strongly stimulated both p46 and p54 JNK MAP
kinases with maximum activity of both isoforms achieved at 30 min
before declining toward basal levels within 60 min in J774 cells (Fig. 1, G and H). Moreover, p46 and p54 JNK were both
stimulated in murine peritoneal macrophages by either LPS or LPS plus
IFN- within 10 min (Fig. 1I). These results clearly show
that LPS stimulates all three classes of MAP kinase in macrophages, but
with differential kinetics of activation.
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A selective and potent inhibitor of the ERK MAP kinase cascade,
PD98059, mediates its effects by binding to and inactivating the
ERK-specific MAP kinase kinase, MEK. It has no effect on any of the
components of the JNK or p38 MAP kinase cascades (40).
Similarly, the compound SB203580 is a selective and potent inhibitor of
p38 MAP kinase, which does not affect either ERK or JNK MAP kinases
(41). Therefore, these reagents are useful pharmacological
tools to identify the functional activities mediated by p38 and ERK MAP
kinases. To ensure that these inhibitors are specific for p38 and MEK
in murine macrophages, we examined the effects of these reagents on the
dual phosphorylation of their targets and heterologous MAP kinases in
J774 cells and BALB/c peritoneal macrophages (Fig. 2 and results not shown). As expected,
PD98059 (20 µM) profoundly inhibited both the basal and stimulated
ERK MAP kinase activation, but had no effect on phosphorylation of p38
or SAPK/JNK (Fig. 2). Indeed, higher concentrations of PD98059
completely abrogated ERK MAP kinase activation (results not shown).
Conversely, SB203580 inhibited p38, but not ERK or JNK phosphorylation
(Fig. 2). Preincubation of cells with SB203580 leads to an increase in
both basal and stimulated ERK (particularly ERK-1) activation,
suggesting that p38 may act, at least in part, to exert inhibitory
effects on ERK signaling in J774 cells. This finding is consistent with
the proposed regulatory cross-talk postulated to occur among the
different MAP kinase signaling cassettes (47).
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To investigate whether ERK or p38 MAP kinases are involved in the
LPS-stimulated induction of iNOS in macrophages, we determined whether
PD98059 or SB203580 affects LPS-stimulated
NO2- production by J774 cells
and murine peritoneal macrophages. Both PD98059 (0–50 µM) and
SB203580 (0–20 µM) inhibited LPS-stimulated
NO2- induction in a
dose-dependent manner (Fig. 3 and results
not shown). At the concentrations used, these compounds did not affect
cell viability (results not shown). Whilst treatment with PD98059 (up
to 50 µM) only partially inhibited
NO2- generation in J774 cells
(Fig. 3A), NO2-
production was almost completely abolished by incubation with 5 µM of
SB203580 (Fig. 3B). These results were confirmed by Western
blot analysis of iNOS expression, which showed that whilst PD98059 (20
µM) only partially blocked the induction of iNOS protein expression
(41%), SB203580 (5 µM) almost completely abrogated LPS-induction of
iNOS expression (Fig. 3C). LPS-stimulated TNF- production
(Northern blot analysis of mRNA and ELISA measurement of secreted
protein) from J774 cells was similarly inhibited by PD980509 and
SB203580 (results not shown). Taken together, these results suggest
that while both p38 and ERK MAP kinases appear to play a positive role
in the LPS-mediated induction of iNOS and TNF- production, p38
activation is more critical and appears to be necessary and sufficient
for effector induction. The physiological relevance of these findings
was underlined by studies which showed that PD98059 (20 µM) and
SB203580 (5 µM) similarly inhibited
NO2- production in murine
peritoneal macrophages resulting from stimulation with either LPS or
LPS plus IFN-. Suppression of
NO2- generation by these MAP
kinase inhibitors was not restricted to a particular haplotype as
essentially identical results were obtained with macrophages derived
from BALB/C (Fig. 3D) and 129 mice (results not shown).
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Stimulation of macrophages with LPS leads to the generation of
IL-12 (p40), which forms the biologically active heterodimeric form of
IL-12 (p70) with the constitutively expressed IL-12 (p35)
(48). To assess the relative roles of ERK and p38 MAP
kinases in LPS-mediated IL-12 induction, we investigated whether
PD98059 or SB203580 affected the LPS-stimulated expression of IL-12
(p40) in J774 macrophages. PD98059 enhanced the production of IL-12
(p40) in a dose-dependent manner (1–40 µM) (Fig. 4A), suggesting that ERK MAP
kinases play a negative regulatory role in the LPS-mediated induction
of p40. In contrast, SB203580 markedly inhibited IL-12 (p40) production
in a dose-dependent manner with an IC50 of about
5 µM (Fig. 4B), indicating that p38 positively transduces
signals in the induction of IL-12 (p40) synthesis. These findings were
corroborated by studies in murine peritoneal macrophages, which showed
that while IL-12 (p40) production was dramatically enhanced by PD98059
(20 µM), it was profoundly suppressed by SB203580 (5 µM; Fig. 4C). To determine whether either of these effects on IL-12
production reflected immunomodulation by the anti-inflammatory
cytokine, IL-10, which has previously been reported to be produced from
LPS-stimulated human monocytes in a p38 MAP kinase-dependent manner
(49), we measured the production of IL-10 from peritoneal
macrophages stimulated with LPS, IFN-, or LPS plus IFN- in the
presence or absence of PD95089 or SB203580. However, we were unable to
detect any IL-10 release from murine peritoneal macrophages (or indeed
from J774 cells) under any of the conditions tested, indicating that
the ERK and p38 MAP kinases did not exert their regulatory effects on
IL-12, NO2-, or TNF- via
generation/suppression of the anti-inflammatory cytokine, IL-10.
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, A and
B), SB203580 delayed and reduced the overall level of IL-12
(p40) mRNA expression. In contrast, neither reagent had any effect on
the levels of ß-actin expression (Fig. 5 and results not shown).
Thus, these data demonstrated that, although LPS stimulates both ERK
and p38 (Fig. 1
), these MAP kinases play opposing roles in the
regulation of signals leading to the induction of IL-12 (p40)
mRNA.
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B binding
to iNOS and IL-12 promoters
The transcription factor NF-B is involved in the regulation of
both iNOS and IL-12 gene induction. Therefore, we investigated whether
the ERK or p38 MAP kinases mediate their effects on
NO2- and IL-12 synthesis by
modulating NF-B activity. LPS stimulated binding of NF-B to the
iNOS and IL-12 promoters (Fig. 6). Ab
supershift assays showed that these binding complexes comprised
p65-p50, c-Rel-p50 complexes, and p50/p50 homodimers (Fig. 6).
Preincubation of the cells with SB203580 or PD98059 did not affect the
binding of these complexes (Fig. 6), suggesting that neither p38 nor
ERK MAP kinase activation appears to be necessary or sufficient for
NF-B binding to the NF-B binding sites of either the iNOS or
IL-12 promoters.
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LPS stimulates the binding of two nuclear complexes (Fig. 7A, bands a and
b) to the IRF-site in the iNOS promoter. Experiments using
SB203580 or PD98059 to probe the role of p38 and ERK MAP kinases in
promoting such complex formation showed that abrogation of p38 MAP
kinase, but not ERK MAP kinase, prevented binding of complex b to the
IRF site on the iNOS promoter (Fig. 7). This is consistent both with
our results above showing a key role for p38 MAP kinase in promoting
iNOS induction (Fig. 3) and with previous reports that IRFs are major
regulatory elements in the induction of iNOS (13, 14, 15). At
present, the components of the LPS-induced IRF-like complexes are
unclear, but Ab supershift studies showed that they do not contain
IRF-1, IRF-2, ICSBP (IFN consensus sequence-binding protein), or ICSAT
(a homologue of mouse Pip) (data not shown).
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B). Moreover, neither SB203580 nor
PD98059 blocked Ets nuclear translocation (Fig. 8), suggesting that neither ERK nor p38
MAP kinases mediated their regulation of IL-12 transcription by
targeting F1 complex formation.
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We have previously shown that a major parasite cell-surface
molecule, phosphoglycan, of Leishmania can selectively
inhibit the synthesis of IL-12 (p40), but not
NO2- or TNF-, by J774
macrophages (Ref. 9 and results not shown). Moreover, this
inhibition of IL-12 could be mimicked by sPG containing the
galactose(ß1–4)mannose(1)-PO4 repeating
units of the parasite molecule (9). Although this
inhibition was at the transcriptional level, it was not mediated
through modulation of NF-B binding and hence was reminiscent of the
ERK MAP kinase-mediated suppression of IL-12 production. Therefore, we
investigated whether sPG modulates IL-12 production by stimulating ERK
MAP kinase in J774 macrophages (Fig. 9).
We found that sPG stimulated ERK activity as evidenced by Western
blotting studies using Abs that recognize only the dually
(Thr/Tyr)-phosphorylated forms of ERK-1 and -2. sPG stimulated both
ERK-1 and -2, and such activation was sustained for at least 30 min
(Fig. 9A). Furthermore, we found that sPG enhanced
LPS-stimulated ERK MAP kinase activity in J774 cells (Fig. 9B), supporting our proposal that sPG may inhibit IL-12
(p40) production in J774 cells by increasing ERK-mediated suppression
of transcription. Evidence that ERK MAP kinase does indeed mediate the
inhibitory effects of sPG was provided by experiments in which the MEK
inhibitor, PD98059, prevented sPG-inhibition of IL-12 production (Fig. 9C).
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(Fig. 10A), which is also subject
to the opposing regulatory signals mediated by ERK and p38 MAP kinases
(Fig. 4C). sPG-mediated inhibition of IL-12 (p40) production
was found to be biologically relevant, as it was reflected by a similar
inhibition of the generation of the biologically active p70 form of
IL-12 (Fig. 10B). Moreover, such sPG-mediated inhibition of
IL-12 production was blocked by the MEK inhibitors PD98059 or U0126
(Fig. 10A and results not shown). Western blot analysis of
the dually phosphorylated forms of ERK-1 and ERK-2 in murine peritoneal
macrophages confirmed that sPG stimulated ERK-1 and ERK-2 activation
under physiological conditions, and the suppressive effects of sPG on
IL-12 production correlated with a substantial enhancement of the ERK
activity observed in response to either LPS or LPS plus IFN- (Fig. 10C). Interestingly, these data also suggested that IFN-
may exert at least some of its synergistic effects on LPS-stimulated
IL-12 production by suppressing the levels of ERK MAP kinase activation
observed in response to LPS alone. However, sPG-mediated suppression of
IL-12 production is not responsible for conferring resistance or
susceptibility of mice to Leishmania infection as
essentially identical results were observed in BALB/c (susceptible;
Fig. 10) and 129 (resistant) mice (results not shown). Moreover, this
suppression of IL-12 does not reflect stimulation of IL-10 by sPG
(results not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production, by subverting the
host’s regulatory mechanism of ERK MAP kinase-mediated suppression of
IL-12 production.
All three classes of MAP kinase, ERK, JNK, and p38, are simultaneously
activated by LPS in macrophages with maximal activation occurring
10–30 min poststimulation in J774 cells (Fig. 1). However, the
individual subtypes exhibit differential kinetics of activation in
response to LPS. For example, while the activation of p46 and p54 JNK
MAP kinases is relatively slow (detectable between 1–10 min) and
returns almost to basal levels within 60 min, LPS-stimulation of ERK-1
and -2 or p38 activation is more rapid (within 1 min) and appears to be
sustained for up to at least 60 min (Fig. 1). Interestingly, studies
using the p38-selective inhibitor, SB203580, show that p38 MAP kinase
acts to suppress ERK-1 and -2 activation in macrophages (Fig. 2), a
finding consistent with an earlier report showing that p38 also
negatively regulated basal and Fc
RI-stimulated activation of ERK-2
in mast cells (47). More importantly, use of this
inhibitor showed that p38 plays an essential role in the LPS-mediated
induction of iNOS (Fig. 3) and promotes the up-regulation of IL-12
(Figs. 4 and 5). In contrast, studies with PD98059, the MEK1 inhibitor
that selectively targets the ERK MAP kinase signaling cascades, showed
that the ERK MAP kinases only contribute partial, nonessential,
positive regulatory signals to the induction of iNOS and act to
suppress expression of IL-12 (p40) (
Figs. 3–5).
Previous studies on the role of individual MAP kinases in the induction
of iNOS have produced conflicting results, suggesting that the
regulation of such gene expression varies in a receptor- and cell
type-dependent manner. For example, while both ERK and p38 were shown
to play positive, but nonessential, regulatory roles in the
LPS-mediated induction of iNOS mRNA in microglia or astrocytes
(33), ERK MAP kinases did not appear to be involved in the
response of rat glioma cells to LPS plus IFN- (50). In
addition, while ERK MAP kinases also appeared to play a positive
regulatory role (51) in transducing the IL-1-mediated
induction of iNOS in a number of systems, PD98059 had no effect on such
NO production in other cell types (52). Similarly, while
p38 activation had also been shown to promote IL-1 stimulation of iNOS
mRNA induction in chondrocytes (53), astrocytes
(52), and pancreatic islets (51), it served
to suppress induction of iNOS mRNA in glomerular mesangial cells
(54). Moreover, while Da Silva et al. (52)
showed that SB203580 inhibited IL-1 stimulated NO production, they
found that it had no effect on NF-B activation in astrocytes,
results consistent with our own findings in macrophages that p38 MAP
kinase plays a key role in LPS-stimulated induction of iNOS without
modulating NF-B binding to the iNOS promoter. Interestingly,
considering that NF-B activation has been proposed to play a key
role in iNOS induction (14), we find that neither p38 nor
ERK MAP kinases appear to play any role in promoting NF-B (p50/p50,
p50/c-rel, and p50/p65 complexes) binding to the iNOS promoter (Fig. 6), despite ERK promoting and p38 MAP kinase being required for iNOS
induction (Fig. 3). In contrast, experiments using SB203580 or PD98059
showed that abrogation of p38, but not ERK, MAP kinases prevented
binding of an IRF-like complex to the iNOS promoter. Although we have
not as yet identified the components of this novel LPS-stimulated
IRF-like complex, we have ruled out a role for any of the
well-characterized factors (IRF-1, IRF-2, ICSBP (IFN consensus
sequence-binding protein), or ICSAT). Taken together, these results not
only suggest that NF-B binding may not be necessary or sufficient
for induction of iNOS but rather support previous studies (including
those using IRF knockout mice) proposing that IRFs are major regulatory
elements in the induction of iNOS (13, 15). Moreover, they
also suggest that p38 and ERK MAP kinases do not play universal roles
in the regulation of iNOS induction but rather transduce differential
regulatory effects that vary in a receptor- and cell type-dependent
manner, perhaps reflecting cell lineage-restricted expression of
isoforms of MAP kinase and/or transcription factors.
Little is known regarding the molecular mechanisms underlying the
induction of biologically active IL-12 apart from the fact that the
transcription factors, NF-B (18) and Ets (17, 55), appear to be important in the LPS-stimulated
transcriptional activation of the IL-12 (p40) subunit. While p50/p65
and p50/c-Rel heterodimers of the NF-B complexes have been reported
to be involved (12), the Ets region interacts
with a nuclear complex termed F1, which includes multiple nuclear
factors (IRF-1, c-Rel, and GLp109) and appears to be highly inducible
by either IFN- or LPS stimulation (17). We found that
neither SB203580 nor PD98059 affect binding of NF-B complexes
(p50/p50, p50/c-rel, and p50/p65 dimers) to the IL-12 promoter (Fig. 6), suggesting that NF-B complexes do not transduce either the p38
promotion of IL-12 induction or the ERK-mediated suppression of IL-12
(p40). This proposal is supported by our findings that while
Leishmania sPG inhibits IL-12 production by stimulating ERK
MAP kinase activity (Figs. 9 and 10), it does not modulate NF-B
binding to the IL-12 promoter (9). Similarly, neither
SB203580 nor PD98059 abrogates F1 complex formation (Fig. 7) or Ets
nuclear translocation (Fig. 8), suggesting that assembly of the F1
complex also does not provide either the positive or negative
regulatory target of MAP kinase action on the IL-12 promoter.
In conclusion, taken that ERK and p38 MAP kinases differentially regulate the LPS-mediated induction of iNOS and IL-12, together with the fact that ERK MAP kinases play opposing roles in modulating iNOS and IL-12 induction, our results suggest that such differential signaling of p38 and ERK MAP kinases may play an important role in determining the type of functional response resulting from stimulation of macrophages with LPS. Many immunomodulatory receptors on macrophages can also differentially signal via one or more MAP kinases. Our finding that distinct subtypes of these signaling cascades make qualitatively different contributions to induction of individual macrophage responses (such as iNOS and IL-12) suggests that macrophages can modify their response to pathogens depending on their environment. Similarly, pathogens may act to evade/modulate the host immune response by subverting host-MAP kinase regulation of macrophage effector responses. This is evidenced by our data demonstrating that Leishmania may suppress resistance to infection by "switching on" ERK MAP kinase-mediated negative regulation of IL-12 production and hence preventing generation of a protective Th1 immune response. That "susceptible" (BALB/c) and "resistant" (129) strains of mice are not differentially sensitive to sPG-mediated inhibition of IL-12 production following in vitro stimulation of macrophages suggests that sPG subversion of ERK MAP kinase-mediated down-regulation of IL-12 is not the molecular rationale underlying susceptibility. However, the ability of sPG to subvert such MAP kinase regulation of IL-12 production does provide the parasite with a mechanism to evade the generation of a protective Th1 immune response and promote parasite survival. Thus, the differential sensitivity of the macrophage effector responses of NO2- and IL-12 to pharmacological modulators of ERK and p38 activation suggests that such reagents could be used as potential immunomodulators to generate the required immunity to combat a particular pathogen or, alternatively, reduce inflammation/pathology associated with certain disease states.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. F.-Y. Liew, Department of Immunology, University of Glasgow, Glasgow G11 6NT, U.K. E-mail address:
3 Abbreviations used in this paper: iNOS, inducible NO synthase; IRF, IFN regulatory factor; MAP, mitogen-activated protein; ERK, extracellular signal-related kinase; JNK, c-jun N-terminal kinase; sPG, synthetic phosphoglycan; SAPK, stress-activated protein kinase; MEK, ERK-specific MAP kinase kinase.
Received for publication May 3, 1999. Accepted for publication September 23, 1999.
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C. Ropert, I. C. Almeida, M. Closel, L. R. Travassos, M. A. J. Ferguson, P. Cohen, and R. T. Gazzinelli Requirement of Mitogen-Activated Protein Kinases and I{{kappa}}B Phosphorylation for Induction of Proinflammatory Cytokines Synthesis by Macrophages Indicates Functional Similarity of Receptors Triggered by Glycosylphosphatidylinositol Anchors from Parasitic Protozoa and Bacterial Lipopolysaccharide J. Immunol., March 1, 2001; 166(5): 3423 - 3431. [Abstract] [Full Text] [PDF]
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T. Matsuguchi, T. Musikacharoen, T. Ogawa, and Y. Yoshikai Gene Expressions of Toll-Like Receptor 2, But Not Toll-Like Receptor 4, Is Induced by LPS and Inflammatory Cytokines in Mouse Macrophages J. Immunol., November 15, 2000; 165(10): 5767 - 5772. [Abstract] [Full Text] [PDF]
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A. B. Carter and G. W. Hunninghake A Constitutive Active MEK right-arrow ERK Pathway Negatively Regulates NF-kappa B-dependent Gene Expression by Modulating TATA-binding Protein Phosphorylation J. Biol. Chem., September 1, 2000; 275(36): 27858 - 27864. [Abstract] [Full Text] [PDF]
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A. S. Kristof, J. Marks-Konczalik, and J. Moss Mitogen-activated Protein Kinases Mediate Activator Protein-1-dependent Human Inducible Nitric-oxide Synthase Promoter Activation J. Biol. Chem., March 9, 2001; 276(11): 8445 - 8452. [Abstract] [Full Text] [PDF]
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C. Zhu, K. Gagnidze, J. H. M. Gemberling, and S. E. Plevy Characterization of an Activation Protein-1-binding Site in the Murine Interleukin-12 p40 Promoter. DEMONSTRATION OF NOVEL FUNCTIONAL ELEMENTS BY A REDUCTIONIST APPROACH J. Biol. Chem., May 18, 2001; 276(21): 18519 - 18528. [Abstract] [Full Text] [PDF]
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