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: Role of the p38 and p42/44 Mitogen-Activated Protein Kinases1
Kennedy Institute of Rheumatology, Hammersmith, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. LPS signal transduction in monocytes has
been shown to involve activation of the p38 and p42 mitogen-activated
protein kinase (MAPK) cascades. The results in this paper indicate that
inhibition of p38 MAPK potently inhibited the production of IL-10,
IL-1ß, and TNF-, whereas blockade of the p42/44 MAPK pathway,
while partially inhibiting TNF- and IL-1ß production, had no
effect on monocyte secretion of IL-10. Furthermore, neither the
inhibition of monocyte TNF- induced by IL-10 nor the stimulation of
soluble TNF receptor production was affected by inhibition of the
p42/44 MAPK pathway, suggesting that this signaling event is not
involved in either monocyte production of or anti-inflammatory
responses to IL-10. These data raise the interesting possibility that
proinflammatory TNF--mediated effects may be selectively blocked
without modulating the induction or the response to IL-10, whereas the
signaling events associated with the anti-inflammatory events
induced by IL-10 remain to be elucidated. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, and IL-6 (3, 4), and down-regulating their capacity to serve as accessory cells for
stimulation of T cell function (3, 4, 5). In addition, IL-10 has been
shown to stimulate monocyte expression of soluble TNF receptors
(sTNFR)5 (6), the
natural inhibitors of TNF-, and to enhance the production of IL-1R
antagonist (IL-1Ra) (7, 8), which competitively inhibits IL-1 binding
to the membrane receptor. In vivo studies have shown that mice become
more sensitive to endotoxic shock following treatment with
anti-IL-10 Abs, while administration of IL-10 suppressed lethal
endotoxemia and reduced serum TNF- (9, 10). Further, IL-10-deficient
mice develop chronic enterocolitis, arising from uncontrolled immune
responses in the bowel, and marked irritant and contact
hypersensitivity responses in the skin (11, 12). As a result of such
anti-inflammatory properties, IL-10 has been suggested as a
therapeutic agent for inflammatory conditions such as rheumatoid
arthritis and inflammatory bowel disease, and clinical trials to test
this hypothesis are in progress.
IL-10 is produced by a variety of immune cell types, including cells of
the monocyte/macrophage lineage (1, 2); nevertheless, little is known
concerning the regulation of its synthesis by monocytes/macrophages. A
number of studies suggest that both soluble factors and cell-cell
contact-mediated signals are involved in the stimulation of monocyte
IL-10 production (13, 14, 15, 16). Notably, several groups have shown that
TNF- plays a role in the induction of IL-10 by stimulated monocytes
(17, 18). To date however, bacterial endotoxin (LPS) has been the most
consistent and best characterized signal for IL-10 production by
monocytic cells. Activation of human monocytes by LPS in vitro results
in the rapid production of proinflammatory cytokines, including
TNF-, IL-1, and IL-6, followed later by the secretion of the
anti-inflammatory IL-10, sTNFR, and IL-1Ra, which down-regulate or
antagonize the proinflammatory mediators. The kinetics of cytokine
production has led to the suggestion that different regulatory
mechanisms are involved in the expression of the pro- and
anti-inflammatory cytokines.
LPS signal transduction in monocytes involves binding to cell surface
CD14 (19) and has been shown to include activation of tyrosine kinases
(20, 21), protein kinase C (22, 23, 24), and the mitogen-activated protein
kinases (MAPK), p38, p42/44(ERK) and p54(stress-activated protein
kinase/JNK) (23, 25, 26). While the role of the MAPKs in LPS-induced
signaling is probably the best characterized, the relationship between
the activation of these signaling molecules and induced cytokine
expression is still obscure. The only exception to this is the
observation that inhibition of the p38 MAPK with specific imidazole
inhibitors (e.g., SB203580) prevents translation of the TNF- mRNA
(25). Activation of the p42/44 MAPK pathway has also been implicated in
TNF- expression, but these studies have been performed in cell lines
transfected with various mutant forms of Raf-1 kinase, a proximal
activator of the p42/44 MAPK pathway (27). There is little information
on the role of either of these kinases in regulating the expression of
other cytokines in monocytes in general, and none regarding IL-10
synthesis in particular. A recent report has suggested that IL-10
production is dependent on protein tyrosine kinase and protein kinase C
activation (28), while several studies suggest that factors that
elevate cAMP are involved in the regulation of monocytic IL-10
production, primarily at the mRNA level (18, 29, 30). In the present
study we have investigated the regulation of monocyte IL-10 production
following stimulation by LPS. The results demonstrate the involvement
of endogenous monocyte-derived IL-1 in LPS-induced IL-10 production and
in addition confirm the importance of endogenous TNF- in the
secretion of IL-10. We further demonstrate that the p38 and p42/44 MAPK
pathways differentially regulate monocyte production of the cytokines
IL-10, IL-1ß, and TNF-. Moreover, data suggest that monocyte
anti-inflammatory responses to IL-10 and monocyte production of
IL-10 do not involve the p42/44 MAPK cascade.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human recombinant cytokines were gifts: TNF- from Prof. W.
Stec (Center of Molecular and Macromolecular Studies, Lodz, Poland),
IL-10 from Dr. S. Smith (Schering Plough, Kenilworth, NJ), IL-6 from
Dr. F. Di Padova (Sandoz, Basel, Switzerland), IL-1ß from Dr. J.
Kenny (Syntex, Palo Alto, CA), and human recombinant IL-1Ra from Dr. A.
Berger (Upjohn Laboratories, Kalamazoo, MI). The IL-1Ra was tested in a
functional assay that measures neutralization of IL-1 activity, as
previously described (31). Salmonella abortus equi LPS was a
gift from Dr. C. Galanos (Max Planck Institute, Freiberg, Germany).
PD98059 MEK1 inhibitor was obtained from Calbiochem-Novabiochem Ltd.
(Nottingham, U.K.). SB203580 was a gift from Dr. J. Lee (SmithKline
Beecham Pharmaceuticals, King of Prussia, PA). Cell culture medium
consisted of RPMI 1640 containing 300 µg/ml L-glutamine
(BioWhittaker, Verviers, Belgium) supplemented with 100 U/ml penicillin
(BioWhittaker), 100 µg/ml streptomycin (BioWhittaker), and 10%
heat-inactivated FCS (Sigma Chemical Co., Poole, U.K.). All monocyte
reagents and culture media were shown to contain <0.1 U/ml endotoxin
as measured using the chromogenic Limulus amebocyte lysate
assay (BioWhittaker).
Antibodies
Whole Ab and Fab of the chimeric mouse Fv/human IgG1,k
anti-TNF- mAb cA2 were gifts from Dr. J. Ghrayeb (Centocor,
Malvern, PA) (32). The high affinity cA2 mAb has previously been shown
to efficiently neutralize TNF- in a number of in vitro bioassays at
concentrations of 1 to 4 µg/ml (32, 33) (data not shown). The
neutralizing anti-human GM-CSF sheep polyclonal Ab Sh7 was a gift
from the Genetics Institute (Cambridge, MA). Anti-human IL-8 mouse mAb
A5.12.14 (IgG2a) was a gift from Genentech, Inc. (South San Francisco,
CA). Neutralizing anti-human IFN-2 mouse mAb MT3/B4 (IgG1),
anti-human IFN- sheep polyclonal Ab H51, and anti-human
IFN-ß sheep polyclonal Ab H60 were gifts from Dr. A. Meager (National
Institute for Biologic Standards and Control, London, U.K.). The
neutralizing anti-human IL-6 sheep polyclonal Ab was a gift from
Dr. L. Aarden (Central Laboratory of The Netherlands Red Cross Blood
Transfusion Service). Isotype control Abs were mouse IgG1 and IgG2a
(anti-TNP; PharMingen, San Diego, CA) and sheep IgG (Sigma
Chemical Co.).
Monocyte purification and culture
Human PBMC were isolated from single donor plateletpheresis
residues purchased from the North London Blood Transfusion Service
(Colindale, U.K.). Mononuclear cells were isolated by Ficoll/Hypaque
centrifugation (specific density, 1.077 g/ml; Nycomed Pharma A.S.,
Oslo, Norway) before separation in a Beckman JE6 elutriator (High
Wycombe, U.K.). Elutriation was performed in culture medium with 1%
heat-inactivated FCS. Monocyte purity was assessed by flow cytometry
using fluorochrome-conjugated anti-CD45, anti-CD3,
anti-CD14, and anti-CD19 mAbs (Becton Dickinson, Oxford, U.K.)
and routinely consisted of >80% CD14-expressing cells, <0.5%
CD19-expressing cells, and <3% CD3-expressing cells. Monocytes were
cultured at 6 x 106 cells/ml in monocyte medium
in flat-bottom 96-well culture plates (Nunc Life Technologies Ltd.,
Paisley, Scotland). At the start of the culture period cells either
were left unstimulated or were cultured with the following reagents as
indicated in the text: LPS (10 ng/ml), IL-1Ra (20 µg/ml), IL-10 (10
ng/ml), whole Ab or Fab of anti-TNF- mAb cA2 (10 µg/ml),
anti-IL-6 polyclonal Ab, anti-GM-CSF polyclonal Ab Sh7,
anti-IFN- polyclonal Ab H51, anti-IFN-ß polyclonal Ab H60,
and the isotype control sheep polyclonal IgG (all at a 1/100 dilution
of stock concentration), anti-IFN-2 mAb MT3/B4 (1/100 dilution
of ascetic fluid), anti-IL-8 mAb A5.12.14, and the isotype-matched
IgG1 and IgG2a control mAb (10 µg/ml). In some experiments monocytes
were treated for 1 h with PD98059 or SB203850 at the indicated
concentrations before stimulation with LPS. After 24-h incubation at
37°C with 5% CO2, culture supernatants (200 µl/well,
three wells per condition) were harvested and stored at -20°C until
used. All experiments were performed at least three times, and the
figures show representative examples of these experiments.
Measurements of cytokines and sTNFR by sandwich ELISA
Reagents for the IL-10 ELISA were gifts from Dr. K. Moore (DNAX,
Palo Alto, CA) and Dr. S. Smith (Schering Plough), and the ELISA was
performed as previously described (34). The range of the assay was from
0.04 to 10 ng/ml. Reagents for the TNF- ELISA were provided by Dr.
W. Buurman (Rijks Universiteit Limburg, Maastricht, The Netherlands).
The ELISA was performed as described previously (35) using the coat
anti-TNF- mAb 61E71 and was developed using a rabbit polyclonal
anti-TNF- Ab. The rabbit polyclonal Ab was detected using a
peroxidase-conjugated goat anti-rabbit IgG(H+L) (Jackson
ImmunoResearch Laboratories, Inc, West Grove, PA) followed by an
appropriate substrate. The range of the assay was from 0.004 to 10
ng/ml. Reagents for the IL-1ß ELISA were purchased from BioSource
(distributed by Lifescreen Ltd., Watford, U.K.) The limit of detection
in this assay is 40 pg/ml. All results are expressed as the mean
concentration of cytokines ± SD obtained per condition. Reagents
for the IL-6 ELISA were gifts from Dr. F. Di Padova (Sandoz, Basel,
Switzerland), and the ELISA was performed using the paired
anti-IL-6 Abs LN314–14 and LN1 14–110 as previously described
(36). The range of the assay was from 0.1 to 10 ng/ml. The sTNFR were
assayed in the cell supernatants by ELISA as previously described (37)
using anti-p75 sTNFR mAb 4C8 as the capture Ab and a rabbit
anti-p75 sTNFR polyclonal Ab as the detection Ab (gifts from Dr. W.
Buurman, Rijks Universiteit Limburg, Maastricht, The Netherlands).
Results are expressed as the mean of triplicate samples, and the
detection limit of the ELISA was 20 pg/ml.
p42 MAPK and MAPKAP-2 kinase assays
Monocytes (3 x 106) were stimulated with LPS (100 ng/ml) for 10 min. Following stimulation, cells were lysed in 400 µl of lysis buffer (20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM ß-glycerol phosphate, 200 mM NaCl, 1% Triton-X-100, 1 mM DTT, 10 mM sodium fluoride, 1 mM sodium ortho-vanadate, 1 mM DTT, 1 mM PMSF, 3 µg/ml aprotinin, 2.5 µg/ml pepstatin, and 1 µg/ml leupeptin). Postnuclear lysates were incubated with 2 µl of rabbit anti-p42 MAPK (Dr. P Cohen, Dundee, U.K.) or 3 µl of anti-MAPKAP-2 (Upstate Biotechnology, Inc., Lake Placid, NY), and 20 µl (50 µl for the MAPKAP-2 kinase assay) of a 50% suspension of protein G in lysis buffer and rotated for 2 h at 4°C. The beads were washed twice in RIPA buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 25 mM ß-glycerol phosphate, 10 mM Na tetra-pyrophosphate, 1 mM sodium ortho-vanadate, 1 mM DTT, 1 mM PMSF, 3 µg/ml aprotinin, 2.5 µg/ml pepstatin, and 1 µg/ml leupeptin) and then twice in assay buffer (20 mM HEPES (pH 7.2), 2 mM EGTA, 10 mM MgCl2, 1 mM DTT, and 0.1 mM sodium ortho-vanadate).
For the p42/44 MAP kinase assay, after the last wash the beads were
left as a 1/1 suspension in assay buffer. Kinase reactions were
conducted at room temperature (under constant agitation) for 20 min
after the addition of 20 µl of 350 µg/ml myelin basic protein
(Calbiochem), 10 µl of 180 µM ATP, and 5 µCi of
[
-32P]ATP. Reactions were stopped by the addition of
25 µl of 4x gel sample buffer and boiled for 3 to 5 min. Samples
were fractionated on a 12.5% SDS-polyacrylamide gel. After the gel had
been fixed in a mixture of water/methanol/acetic acid (5/4/1) and then
dried, the phosphorylated myelin basic protein was detected by
autoradiography using Hyperfilm MP (Amersham, Aylesbury, U.K.).
For the MAPKAP-2 kinase assay, after the last wash the beads were
resuspended in 50 µl of kinase assay buffer containing 30 µM (final
concentration) heat shock protein-27 peptide sequence KKLNRTSVA, (38).
Reactions were initiated by the addition of 10 µl of 180 µM ATP
containing [-32P]ATP (0.5 µCi; Amersham). The
reaction was allowed to proceed for 20 min before termination. This was
achieved by spotting the assay mixture onto squares of p81 paper and
then placing them in 0.75% ortho-phosphoric acid. The squares were
washed three times in the acid and once in acetone before scintillation
counting.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS is a commonly used stimulus for monocyte cytokine production
in vitro, inducing the production of cytokines, including IL-10, IL-1,
TNF-, and IL-6. Monocyte IL-10 is detectable 8 h following LPS
stimulation, with peak production detected at 24 h and maintained
to at least 96 h (4). In contrast, monocyte production of
proinflammatory cytokines such as TNF-, IL-1, and IL-6 is detected
within 2 h of LPS stimulation. We wished to investigate whether
the delayed stimulation of monocyte IL-10 production by LPS was
mediated by such endogenous inflammatory cytokines. To this end,
monocytes were cultured with LPS in the presence or the absence of
neutralizing concentrations of Abs or antagonists to monocyte-derived
cytokines. A representative experiment is shown in Figure 1
, where both anti-TNF- mAb (cA2)
and IL-1Ra potently inhibited LPS-induced monocyte IL-10 production in
a dose-dependent manner. In a series of 10 experiments illustrated in
Figure 1, a maximally neutralizing concentration of cA2 (Centocor,
Malvern, PA) (10 µg/ml) resulted in 55 ± 9.4% inhibition of
LPS-induced IL-10 production. A maximally neutralizing concentration of
IL-1Ra (10 µg/ml) resulted in 52 ± 6% inhibition of IL-10
production. Addition of both cA2 and IL-1Ra to monocyte cultures
enhanced (62 ± 3.8%) the inhibition observed with either
inhibitor alone, but was routinely less than additive. It was noted,
however, that neutralizing concentrations of cA2 and IL-1Ra added
together did not completely abrogate LPS-stimulated IL-10
production.
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2, or isotype-matched control
Abs had no effect on monocyte IL-10 production (data not shown),
indicating that the endogenous cytokines IL-1 and TNF- play a unique
role in LPS-induced IL-10 production. A Fab preparation of the
anti-TNF- mAb also markedly inhibited monocyte IL-10 production,
demonstrating that the effect of anti-TNF- Ab is specific and
not mediated by ligation of monocyte Fc receptors (data not shown).
Addition of exogenous rTNF- or IL-1 and IL-1ß, either alone or
in combination, to monocyte cultures did not induce IL-10 production
unless the monocytes also were primed with LPS.
p42 MAPK and MAPKAP-2 kinase assays: demonstration of inhibitor specificity
There is strong evidence that LPS, IL-1, and TNF- all activate
the p42/44 MAP kinase pathway in a number of cell types (39), and it
was therefore of interest to investigate whether p42/44 MAP kinase
activation is required for the LPS-mediated induction of monocyte IL-10
production. PD98059 is a specific inhibitor of MEK1 (MAPKK), the dual
specificity kinase that activates p42/44 MAPK via phosphorylation on
both threonine and tyrosine residues (40). Inhibition of MEK1 by
PD98059 prevents activation of p42/44 MAPK and the subsequent
phosphorylation of MAPK substrates (41). In Figure 2A, we show that PD98059
inhibits LPS-induced p42 MAPK activation in human monocytes in a
dose-dependent manner, with an IC50 between 1 and 10 µM.
This is in agreement with the reported in vitro MEK inhibitory activity
of PD98059 (41). In contrast, PD98059 at 50 µM had no effect on p38
MAPK activity as demonstrated in Figure 2B, whereas
LPS-induced p38 MAPK activity, as measured by activation of its
substrate kinase (MAPKAP-2), was inhibited to background levels by the
bicyclic imidazole SB203580 (10 µM).
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Monocytes were treated for 1 h with PD98059 before
stimulation with LPS, and the culture supernatants were harvested at
both 4 and 24 h and then assayed for the presence of TNF, IL-1,
and IL-10. Significant levels of all three cytokines were only detected
after 24 h in culture; therefore, only the results from the
24 h points are illustrated. Furthermore, monocytes that were left
unstimulated (no LPS) did not produce detectable amounts of any
cytokine (TNF-, IL-1, or IL-10) at any time point examined,
indicating that adherence to plastic is not sufficient to activate the
relevant pathways to initiate cytokine production.
PD98059 (0.1 to 50 µM) inhibited LPS-induced TNF- and IL-1ß in a
dose-dependent manner, although complete inhibition of either cytokine
was not seen in any experiment. In contrast, PD98059 did not inhibit
LPS-induced IL-10 production at any dose from 0.1 to 50 µM. A
representative experiment is shown in Figure 3, A, B, and
C. In additional experiments, it was noted that PD98059
partially inhibited LPS-induced production of IL-6 in a dose-dependent
manner, with an IC50 of 5 µM (data not shown).
Concentrations of PD98059 >50 µM were not used, as such
concentrations reduced monocyte viability (as determined by exclusion
of 1% nigrosin). Addition of DMSO vehicle alone did not inhibit the
production of any cytokine (data not shown). While the addition of PD
98059 has no effect on IL-10 synthesis (Fig. 3C),
IL-10 levels were reduced when TNF or IL-1 activity was blocked by the
further addition of neutralizing concentrations of cA2 (10 µg/ml) or
IL-1Ra (10 µg/ml; Fig. 3D). Thus, LPS-stimulated
monocytes produced 475 ± 49 pg/ml of IL-10 and were unaffected by
the addition of PD98059 (455 ± 47 pg/ml; not significant), but
IL-10 production was reduced in the presence of cA2 (156 ± 18
pg/ml; p < 0.01) or IL-1Ra (255 ± 28 pg/ml;
p < 0.01) alone and was reduced to almost background
levels when cA2 and IL-1Ra were included together in the presence of
PD98059 (54 ± 4 pg/ml; p < 0.001).
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(3, 4). Addition of 10 ng/ml IL-10 to LPS-stimulated
monocyte cultures resulted in >90% inhibition of TNF- production
as illustrated in Figure 3
A; however, treatment of monocytes
with PD98059 (1–50 µM) before addition of IL-10 did not reverse this
effect, suggesting that the p42/44 MAPK pathway is not involved in this
IL-10-mediated anti-inflammatory effect. Furthermore, IL-10-induced
release of the native inhibitor of TNF, sTNFR (6), was not blocked by
the inclusion of PD98059 at concentrations ranging from 1 to 50 µM in
a total of three experiments (Fig. 4A), whereas
LPS-induced p75 sTNFR release (Fig. 4B) was inhibited
in a dose-dependent manner by the p42 MAPK inhibitor.
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It has previously been demonstrated (42) that the pyridinyl
imidazaole compounds that inhibit p38 MAPK inhibit LPS-induced TNF and
IL-1 production. We sought to determine whether LPS-induced IL-10
production was likewise inhibited. Treatment of monocytes for 1 h
with SB203580 resulted in a marked dose-dependent inhibition of
LPS-induced IL-10 production, with concentrations >5 µM reducing
IL-10 levels to below the minimum sensitivity of the ELISA. A
representative experiment is shown in Figure 5, where SB203580 induced dose-dependent
down-regulation of IL-10 production, with an IC50 of
44 nM. We also confirmed the potent dose-dependent inhibition of both
IL-1ß and TNF- production, with IC50 values of 35 and
70 nM, respectively, in agreement with published results (42). We
noted, however, that there was some variation in IC50
values for SB203580 inhibition between experiments. This variation may
be related to the use of primary monocytes rather than cell lines, with
different donors having different sensitivities to the drug or
different responses to the LPS activation signal.
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(10
ng/ml), either alone or together, after pretreatment of monocytes with
SB 203580. Figure 6 illustrates that
LPS-induced IL-10 production in this experiment was enhanced by the
further addition of rTNF-/IL-1ß. However, IL-10 synthesis was once
again inhibited in a dose-dependent manner by the addition of the p38
inhibitor, SB 203580, and this blockade was not rescued by the addition
of TNF- or IL-1 alone or together. Taken together, this suggests
that the p38 MAPK pathway is involved in IL-10 synthesis, which itself
is both dependent on and independent of IL-1 and TNF- synthesis.
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). As shown
above, 10 µM PD98059 did not inhibit LPS-induced IL-10, and in
addition, PD98059 did not enhance SB203580-mediated inhibition of IL-10
production at any concentration of SB203580 tested (0.016 to 50 µM).
In contrast, PD98059 induced marked inhibition of LPS-stimulated
TNF- production and had an additive effect with SB203580 to inhibit
TNF- production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in LPS-stimulated monocyte IL-10
production. As IL-10 inhibits monocyte production of IL-1 and TNF-,
it is apparent that these three cytokines form an autoregulatory
feedback loop.
Addition of a combination of anti-TNF- mAb cA2 and IL-1Ra to
LPS-stimulated monocyte cultures did not markedly enhance the
inhibition caused by either cA2 or IL-1Ra alone, suggesting that IL-1
and TNF- regulate IL-10 production by a common mechanism. Previous
studies have suggested that the TNF- primarily regulates IL-10
transcription, and it is of interest that the human IL-10 promoter
contains an activating protein-1 recognition site (18), as both IL-1
and TNF- can induce activating protein-1 (43, 44). However, further
investigation of IL-10 production at the levels of transcription,
translation, and secretion is necessary to determine the precise roles
of IL-1 and TNF- in these processes.
The involvement of both endogenous IL-1 and TNF- in monocyte IL-10
production was not unexpected, as these two cytokines have a similar
broad range of physiologic effects. A previous report has suggested
that IL-1 did not regulate IL-10 production (17); however, in those
studies either an anti-IL-1 Ab or an anti-IL-1ß Ab, but
not a combination, was used, and it may not have sufficiently
neutralized the high levels of endogenous IL-1 that monocytes are
capable of secreting. In the present study IL-1Ra was used to prevent
the effects of both IL-1 and IL-1ß. We also noted that addition of
LPS-free, exogenous recombinant IL-1, IL-1ß, or TNF- (in
contrast to previous studies (17)) did not directly induce
LPS-stimulated monocyte IL-10 production (data not shown), although on
some occasions these cytokines did augment LPS-induced IL-10 synthesis
(Fig. 6). It is possible that the high levels of endogenous cytokines
present in the cultures maximally stimulate monocytes, and addition of
exogenous cytokines has no further effect. Several studies, including
the present work, suggest that monocyte cytokine production is
regulated in an autocrine/paracrine network of stimulatory and
inhibitory cytokines such as IL-1, TNF-, and IL-10 (17, 18, 28).
This suggests the existence of both early common and late specific
signal transduction events. The latter may be mediated at least partly
by LPS-induced mediators such as IL-1 and TNF-. The data shown here
also indicate that IL-1/TNF--independent mechanisms of LPS-mediated
IL-10 production exist, as IL-10 was routinely detected in cultures
treated with maximally neutralizing concentrations of IL-1Ra in
combination with anti-TNF- mAb. Further, addition of IL-1,
IL-1ß, and/or TNF- to monocytes in the absence of LPS did not
induce IL-10 production (data not shown). This LPS-induced signal may
involve additional endogenous cytokines, inducible contact-mediated
signals between monocytes, or a distinct biochemical pathway. Indeed,
we have recently demonstrated that TCR-activated T lymphocytes can
induce monocyte IL-10 production in a contact-dependent,
LPS-independent manner (16).
The signaling pathways involved in LPS-stimulated monocyte production of proinflammatory or anti-inflammatory cytokines are not yet fully understood. LPS has previously been shown to activate all three (known) mammalian MAPK pathways, p42/44 (ERK1/2), p38, and p54/JNK (stress-activated protein kinases), in monocytes or macrophages (23, 25, 26). Until recently, p42/44 was the only cloned and well-characterized mammalian MAPK; however, the discovery of two other MAP kinases, p38 and p54, revealed the existence of parallel MAPK cascades that can be activated individually and simultaneously (39, 42, 45), suggesting independent signaling roles for these MAPK cascades.
The data presented here demonstrate that the p42/44 MAPK pathway is not
involved in LPS-stimulated monocyte IL-10 production. LPS-stimulated
TNF- and IL-1ß production was partially inhibited by PD98059, an
inhibitor of MEK1, indicating that these proinflammatory cytokines are
to some extent dependent on the p42/44 MAPK pathway. The PD98059
inhibitor abrogated p42/p44 activity, as illustrated in Figure 2A, with an IC50 between 1 and 10 µM, whereas
the IC50 of this drug for inhibition of TNF- and IL-1ß
was slightly higher (
10 µM; Fig. 3, A and
B). This most likely reflects the difference between
the kinetics of drug activity in the in vitro kinase assay (cells
harvested after 10 min) compared with those in the biologic assay
(cells harvested after 18 h). However, it cannot be excluded that
the PD98059 inhibitor has a target other than MEK, the upstream kinase
in the p42/44 pathway, although to date there is no evidence for
nonspecificity of this drug (55). Furthermore, a number of recent
reports have shown that IL-1 and TNF- activate p42/44 MAPK in a
variety of cell types, including fibroblasts, vascular endothelial
cells, and chondrocytes (39), and in a recent study it was demonstrated
that cross-linking Fc receptors on mouse macrophages results in
p42/44 MAPK activation and TNF- production, which was inhibited by
the PD98059 drug (46). However, the lack of effect of PD98059 on
monocyte IL-10 production suggests that LPS-induced IL-10 synthesis is
not mediated by a p42/44-mediated pathway, although IL-10 synthesis was
reduced in those LPS-stimulated monocyte cultures pretreated with
PD98059, if additionally all TNF- and IL-1 activities were
neutralized using cA2 and IL-1Ra, similar to that seen with cA2 and
IL-1Ra alone (data not shown). This indicates that IL-10 production is
independent of the p42/44 MAPK pathway, but that endogenous TNF and
IL-1 clearly play a role in the IL-10 synthetic pathway.
Despite the wealth of papers demonstrating the importance of IL-10 as
an immunoregulator, the mechanisms by which it exerts these effects
remain poorly understood. To date, IL-10 has been shown to activate two
distinct pathways, the JAK/STAT pathway (47, 48) and the
phosphatidylinositol 3-kinase/S6 kinase pathway (49). However, neither
of these pathways has been shown to mediate the anti-inflammatory
actions of IL-10. We have shown that the p42/44 MAPK pathway is not
involved in IL-10 synthesis in monocytes, and it was of interest to
determine whether IL-10-mediated signaling events in monocytes were
likewise independent of this kinase. Results show that neither
IL-10-induced inhibition of TNF- production nor stimulation of p75
sTNFR was affected by PD98059 inhibitor, thus indicating that the
p42/44 MAPK pathway is not involved in these IL-10-mediated
anti-inflammatory effects. In contrast to the IL-10 effects, the
induction of p75 sTNFR by LPS was sensitive to the p42/44 inhibitor. In
keeping with the cellular data, which indicate that IL-10-induced
anti-inflammatory effects are not mediated by the p42/44 MAPK
pathway, we have shown that IL-10 does not induce MAP/ERK
phosphorylation (as assessed by gel retardation) and hence did not
activate p42 MAPK (data not shown). Furthermore, LPS-induced p42 MAPK
activation, as measured by in vitro kinase assay and by gel
retardation, was not inhibited by IL-10 (data not shown). This is in
contrast to an earlier report (50) in which IL-10 was reported to
inhibit LPS-induced tyrosine phosphorylation of
p56lyn and p56vav, with a
subsequent decrease in Ras and p42 MAPK activation. In addition, IL-10
had no effect on LPS-induced activation of either p38 or p54 MAPK (data
not shown), as assessed using an in vitro kinase assay as measured by
[-32P]ATP incorporation into the relevant substrate
(activating transcription factor-2 or myelin basic protein). The
inability of IL-10 to modify p38 activity was surprising, as this
enzyme is clearly a strong candidate for IL-10 deactivation because it
has been shown to be critically involved in TNF production. IL-10 also
did not inhibit LPS-induced tyrosine phosphorylation of p38 MAPK, nor
was the activity of MAPKAP-2 (a downstream substrate for activated p38
MAPK) inhibited by IL-10 (data not shown). Thus, despite the importance
of IL-10 as a potent immunoregulatory cytokine, the signaling events
associated with the anti-inflammatory effects remain to be
elucidated.
p38 MAPK has previously been shown to be activated by LPS, IL-1, and
TNF- (42). In this report we show for the first time that inhibition
of p38 MAPK with SB203580 results in complete abrogation of LPS-induced
IL-10 production. In addition, we confirm that SB203580 inhibits
LPS-induced TNF-, IL-1ß, and IL-6, in agreement with earlier
studies using related bicyclic imidazoles (25, 51, 52). These results
suggest that activation of the p38 MAPK cascade is an early common
signal necessary for LPS-stimulated monocyte cytokine production. While
the signaling events leading to IL-10 synthesis are complex, we
hypothesize that IL-10 production requires at least two signals; the
first is provided by LPS (or its physiologic equivalent), and the
second by endogenous TNF- and/or IL-1. The fact that the addition of
exogenous rTNF (10 ng/ml) or IL-1ß (10 ng/ml), either alone or
together, failed to rescue IL-10 synthesis in those LPS-stimulated
cultures that had been pretreated with SB203580 indicates that IL-10
production could be dependent upon p38 MAPK at different levels in
terms of endogenous TNF-/IL-1 synthesis as well as at the
LPS-induced and/or TNF-/IL-1 signaling level.
Inhibition of the p42/44 MAPK cascade did not augment the SB203580
inhibition of IL-10 production, providing further evidence that p42/44
MAPK is not involved in LPS-induced IL-10. In contrast, both PD98059
and SB203580 inhibited LPS-induced TNF- singly and acted additively
to reduce monocyte TNF-. The Ras/Raf/ERK kinase pathway has been
suggested to be primarily responsible for LPS-induced transduction of
the TNF- gene (53). The partial inhibition of TNF- production
induced by PD98059 observed in the present study might suggest that
several parallel pathways regulate TNF- gene transcription, and that
these alternative pathways may be sufficient to stimulate the TNF-
promoter despite effective blockade of the p42 MAPK cascade. The
LPS-induced signal leading to TNF- production is believed to branch,
ultimately yielding both transcriptional and translational activation
of TNF- biosynthesis (54). Our data would support this view, as a
combination of PD98059, leading to transcriptional block of TNF-
synthesis, and SB203580, leading to translational block of TNF-
synthesis, resulted in complete abrogation of TNF- production,
suggesting that the p38 and p42/44 MAPK pathways are both necessary for
LPS-induced monocyte TNF- production.
In conclusion, the results demonstrate the different effects of the
MAPK inhibitors on monocyte production of IL-1, TNF-, and IL-10,
indicating that at least two MAPK cascades are involved in
LPS-stimulated production of these cytokines. Further, the differential
susceptibility of pro- vs anti-inflammatory production to PD98059
or SB203580 indicate the differing importance of p42/44 and p38 MAPK in
the signaling events leading to the production of a given cytokine.
TNF- and IL-1 are considered to be key mediators in many
inflammatory conditions, including arthritis and inflammatory bowel
disease, whereas IL-10 is believed to play an important
anti-inflammatory role. Clearly, there is great therapeutic
potential for any compound that interferes with the production or
action of TNF- or IL-1 but does not modulate either the production
of IL-10 or the anti-inflammatory effects mediated by IL-10.
|
Acknowledgments |
|---|
, Dr. A. Berger (Upjohn
Laboratories, Kalamazoo, MI) for the IL-1Ra, Dr. J. Kenny
(Syntex, Palo Alto, CA) for the IL-1ß ELISA reagents, Dr.
F. Di Padova (Sandoz, Basel, Switzerland) for the IL-6 ELISA
reagents, Dr. W. Buurman (Rijks Universiteit Limburg,
Maastricht, The Netherlands) for the TNF- and the sTNFR ELISA
reagents, and Dr. K. Moore (DNAX, Palo Alto, CA) and
Dr. S. Smith (Schering Plough, Kenilworth, NJ) for the
reagents for the IL-10 ELISA. We also thank Dr. C. Galanos
(Max Planck Institute, Freiberg, Germany) for the LPS, Dr. J.
Lee (SmithKline Beecham Pharmaceuticals, King of Prussia, PA) for the
SB203580 inhibitor of p38 MAP kinase, Dr. J. Ghrayeb
(Centocor, Malvern, PA) for the cA2 anti-TNF- mAb, Dr.
A. Meager (National Institute for Biologic Standards and Control,
London, U.K.) for the anti-IFN- and -ß Abs, Dr. L.
Aarden (Central Laboratory of The Netherlands Red Cross Blood
Transfusion Service) for the anti-IL-6 Ab, and Dr. P.
Cohen, Dundee, U.K.) for the anti-p42 MAP kinase Ab. |
Footnotes |
|---|
2 These authors contributed equally to this work.
3 Current address: Department of Medical Microbiology, Sherman Fairchild Science Building, Stanford University School of Medicine, Stanford, CA 94305-5402.
4 Address correspondence and reprint requests to Dr. Fionula M. Brennan, Kennedy Institute of Rheumatology, 1 Aspenlea Road, London, United Kingdom W6 8LH. E-mail address:
5 Abbreviations used in this paper: sTNFR, soluble tumor necrosis factor receptor; IL-1Ra, interleukin-1 receptor antagonist; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein ERK kinase; GM-CSF, granulocyte-macrophage colony-stimulating factor; MAPKAP, mitogen-activated protein kinase-activated protein kinase; cA2, chimeric anti-TNF Ab, A2.
Received for publication February 19, 1997. Accepted for publication September 30, 1997.
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X. Ma, J. Sun, E. Papasavvas, H. Riemann, S. Robertson, J. Marshall, R. T. Bailer, A. Moore, R. P. Donnelly, G. Trinchieri, et al. Inhibition of IL-12 Production in Human Monocyte-Derived Macrophages by TNF J. Immunol., February 15, 2000; 164(4): 1722 - 1729. [Abstract] [Full Text] [PDF]
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G.-J. Feng, H. S. Goodridge, M. M. Harnett, X.-Q. Wei, A. V. Nikolaev, A. P. Higson, and F.-Y. Liew Extracellular Signal-Related Kinase (ERK) and p38 Mitogen-Activated Protein (MAP) Kinases Differentially Regulate the Lipopolysaccharide-Mediated Induction of Inducible Nitric Oxide Synthase and IL-12 in Macrophages: Leishmania Phosphoglycans Subvert Macrophage IL-12 Production by Targeting ERK MAP Kinase J. Immunol., December 15, 1999; 163(12): 6403 - 6412. [Abstract] [Full Text] [PDF]
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A. Aicher, G. L. Shu, D. Magaletti, T. Mulvania, A. Pezzutto, A. Craxton, and E. A. Clark Differential Role for p38 Mitogen-Activated Protein Kinase in Regulating CD40-Induced Gene Expression in Dendritic Cells and B Cells J. Immunol., December 1, 1999; 163(11): 5786 - 5795. [Abstract] [Full Text] [PDF]
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D. L. Foss, M. J. Zilliox, and M. P. Murtaugh Differential Regulation of Macrophage Interleukin-1 (IL-1), IL-12, and CD80-CD86 by Two Bacterial Toxins Infect. Immun., October 1, 1999; 67(10): 5275 - 5281. [Abstract] [Full Text] [PDF]
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D. Stoiber, P. Kovarik, S. Cohney, J. A. Johnston, P. Steinlein, and T. Decker Lipopolysaccharide Induces in Macrophages the Synthesis of the Suppressor of Cytokine Signaling 3 and Suppresses Signal Transduction in Response to the Activating Factor IFN-{gamma} J. Immunol., September 1, 1999; 163(5): 2640 - 2647. [Abstract] [Full Text] [PDF]
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T. van der Bruggen, S. Nijenhuis, E. van Raaij, J. Verhoef, and B. Sweder van Asbeck Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Production by Human Monocytes Involves the Raf-1/MEK1-MEK2/ERK1-ERK2 Pathway Infect. Immun., August 1, 1999; 67(8): 3824 - 3829. [Abstract] [Full Text] [PDF]
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 J. Bondeson, B. Foxwell, F. Brennan, and M. Feldmann Defining therapeutic targets by using adenovirus: Blocking NF-kappa B inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators PNAS, May 11, 1999; 96(10): 5668 - 5673. [Abstract] [Full Text] [PDF]
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K. Miura, J. T. Schroeder, W. C. Hubbard, and D. W. MacGlashan Jr. Extracellular Signal-Regulated Kinases Regulate Leukotriene C4 Generation, But Not Histamine Release or IL-4 Production from Human Basophils J. Immunol., April 1, 1999; 162(7): 4198 - 4206. [Abstract] [Full Text] [PDF]
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K. K. Hale, D. Trollinger, M. Rihanek, and C. L. Manthey Differential Expression and Activation of p38 Mitogen-Activated Protein Kinase {alpha}, {beta}, {gamma}, and {delta} in Inflammatory Cell Lineages J. Immunol., April 1, 1999; 162(7): 4246 - 4252. [Abstract] [Full Text] [PDF]
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J. Bondeson, K. A. Browne, F. M. Brennan, B. M. J. Foxwell, and M. Feldmann Selective Regulation of Cytokine Induction by Adenoviral Gene Transfer of I{kappa}B{alpha} into Human Macrophages: Lipopolysaccharide-Induced, But Not Zymosan-Induced, Proinflammatory Cytokines Are Inhibited, But IL-10 Is Nuclear Factor-{kappa}B Independent J. Immunol., March 1, 1999; 162(5): 2939 - 2945. [Abstract] [Full Text] [PDF]
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K. E. Noble, R. G. Wickremasinghe, C. DeCornet, P. Panayiotidis, and K. L. Yong Monocytes Stimulate Expression of the Bcl-2 Family Member, A1, in Endothelial Cells and Confer Protection Against Apoptosis J. Immunol., February 1, 1999; 162(3): 1376 - 1383. [Abstract] [Full Text] [PDF]
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P. H. Schafer, L. Wang, S. A. Wadsworth, J. E. Davis, and J. J. Siekierka T Cell Activation Signals Up-Regulate p38 Mitogen-Activated Protein Kinase Activity and Induce TNF-{alpha} Production in a Manner Distinct from LPS Activation of Monocytes J. Immunol., January 15, 1999; 162(2): 659 - 668. [Abstract] [Full Text] [PDF]
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J. L. E. Dean, M. Brook, A. R. Clark, and J. Saklatvala p38 Mitogen-activated Protein Kinase Regulates Cyclooxygenase-2 mRNA Stability and Transcription in Lipopolysaccharide-treated Human Monocytes J. Biol. Chem., January 1, 1999; 274(1): 264 - 269. [Abstract] [Full Text] [PDF]
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D. D. Browning, N. D. Windes, and R. D. Ye Activation of p38 Mitogen-activated Protein Kinase by Lipopolysaccharide in Human Neutrophils Requires Nitric Oxide-dependent cGMP Accumulation J. Biol. Chem., January 1, 1999; 274(1): 537 - 542. [Abstract] [Full Text] [PDF]
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P. A. Scherle, E. A. Jones, M. F. Favata, A. J. Daulerio, M. B. Covington, S. A. Nurnberg, R. L. Magolda, and J. M. Trzaskos Inhibition of MAP Kinase Kinase Prevents Cytokine and Prostaglandin E2 Production in Lipopolysaccharide-Stimulated Monocytes J. Immunol., November 15, 1998; 161(10): 5681 - 5686. [Abstract] [Full Text] [PDF]
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K. Rutault, C. A. Hazzalin, and L. C. Mahadevan Combinations of ERK and p38 MAPK Inhibitors Ablate Tumor Necrosis Factor-alpha (TNF-alpha ) mRNA Induction. EVIDENCE FOR SELECTIVE DESTABILIZATION OF TNF-alpha TRANSCRIPTS J. Biol. Chem., February 23, 2001; 276(9): 6666 - 6674. [Abstract] [Full Text] [PDF]
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W. Ma, W. Lim, K. Gee, S. Aucoin, D. Nandan, M. Kozlowski, F. Diaz-Mitoma, and A. Kumar The p38 Mitogen-activated Kinase Pathway Regulates the Human Interleukin-10 Promoter via the Activation of Sp1 Transcription Factor in Lipopolysaccharide-stimulated Human Macrophages J. Biol. Chem., April 20, 2001; 276(17): 13664 - 13674. [Abstract] [Full Text] [PDF]
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J.-i. Okano and A. K. Rustgi Paclitaxel Induces Prolonged Activation of the Ras/MEK/ERK Pathway Independently of Activating the Programmed Cell Death Machinery J. Biol. Chem., May 25, 2001; 276(22): 19555 - 19564. [Abstract] [Full Text] [PDF]
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S. Bechoua and L. W. Daniel Phospholipase D Is Required in the Signaling Pathway Leading to p38 MAPK Activation in Neutrophil-like HL-60 Cells, Stimulated by N-Formyl-methionyl-leucyl-phenylalanine J. Biol. Chem., August 17, 2001; 276(34): 31752 - 31759. [Abstract] [Full Text] [PDF]
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Y.-Z. Wang, P. Zhang, A. B. Rice, and J. C. Bonner Regulation of Interleukin-1beta -induced Platelet-derived Growth Factor Receptor-alpha Expression in Rat Pulmonary Myofibroblasts by p38 Mitogen-activated Protein Kinase J. Biol. Chem., July 14, 2000; 275(29): 22550 - 22557. [Abstract] [Full Text] [PDF]
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