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INTRODUCTION |
Monocyte activation by T cells, such as occurs in autoimmune
inflammatory disease, involves the influence of both cell
contact-dependent as well as cytokine-generated signals
(1). CD40, a member of the tumor necrosis factor receptor superfamily,
and its ligand, CD154, have been identified as a receptor:ligand pair
which contributes to contact-dependent signaling between
these cell types. Early studies of CD40 focused on its role in
co-stimulation of B cell proliferation and immunoglobulin isotype
switching (2), culminating with the finding that X-linked Hyper-IgM
Syndrome, an immunodeficiency characterized by absence of circulating
IgG and IgA and by the absence of germinal centers, is a result of
defects in the gene encoding CD154 (3, 4). A broader role of CD40
signaling was revealed through the finding that CD40 is expressed on
numerous cell types including, in addition to monocytes, dendritic
cells, fibroblasts, keratinocytes, endothelial cells, and vascular
smooth muscle cells (5-11). Stimulation of these cell types through
CD40 induces cell functions that contribute to inflammatory responses, such as activation of cytokine synthesis and enhancement of
co-stimulatory and adhesion molecule expression (12). In
monocytes/macrophages the interaction of CD40 with CD154 has been shown
to result in the activation of inflammatory cytokine production (5, 6) and nitric oxide production (13, 14), as well as rescue from apoptosis
(15, 16). The contribution of CD40 signaling to T cell activation of
macrophages was further substantiated by the demonstration that
CD4+ T cells from CD154-knockout mice are deficient in
their ability to induce macrophage effector function (14). The finding
that humans (17) and mice (18) with defective CD154 genes display an
increased susceptibility to disseminated infections by microorganisms usually contained by cell-mediated immune function also indicates that
productive T cell-macrophage interactions depend on the presence of
functional CD154.
The role of monocyte/macrophage-derived inflammatory cytokines in
inflammatory autoimmune diseases has been well established (19).
Therefore, the demonstration that the CD40-CD154 interaction contributes to the ability of T cells to activate monocyte/macrophage inflammatory cytokine synthesis suggested that this receptor:ligand pair may contribute to the maintenance and/or exacerbation of autoimmune inflammatory disease. In murine models of autoimmune disease, including collagen-induced arthritis, thyroiditis, and experimental autoimmune encephalomyelitis, blockade of the CD40-CD154 interaction blocked development of these autoimmune diseases (20-22). The role of CD154-CD40 interactions in disease onset in these cases has
been ascribed to the requirement for CD40-mediated induction of
co-stimulatory molecules necessary for T cell activation (23, 24).
However, current evidence indicates that the role of the CD154-CD40
interaction goes beyond the initial contact signaling events. For
example, although anti-CD154 treatment inhibits the development of
experimental autoimmune encephalomyelitis, it was also shown that
anti-CD154 treatment of animals after onset of experimental
autoimmune encephalomyelitis reduced the extent and severity of lesions
by more than 50% (22). In addition, obstruction of inflammatory
cytokine signaling can reduce symptoms of ongoing inflammatory disease
as shown recently in clinical studies in which blockade of
TNF1 responsiveness through
treatment with soluble TNF receptor reduced symptoms of rheumatoid
arthritis (25). Clearly, the reduced inflammation resulting from
blockade of CD154-CD40 interactions is due to the inhibition of a
number of crucial outcomes of CD40 signaling, including T cell
induction of monocyte/macrophage inflammatory cytokine synthesis.
Previous work from our laboratory has demonstrated that activation of
monocyte inflammatory cytokine synthesis through the CD154-CD40
interaction is effectively inhibited by the T helper type 2-derived
cytokines IL-4 and IL-10. Numerous studies, in vitro, have
shown that IL-4 and IL-10 down-regulate monocyte/macrophage inflammatory function in response to stimulation with
lipopolysaccharide (LPS) (26-28). In vivo studies have
demonstrated that these cytokines can reduce autoimmune inflammatory
disease (29-33) and recently clinical trials involving use of IL-10 as
therapy for autoimmune disease have been initiated with success (34,
35). Our previous in vitro studies suggest that the in
vivo effectiveness of these cytokines in autoimmune disease may
result, in part, from their direct effect on monocyte/macrophage
activation by T cells mediated through CD40 signaling.
The signaling pathway(s) involved in CD40-mediated induction of
monocyte inflammatory cytokine synthesis have not been fully characterized, nor has the mechanism of IL-4 or IL-10 inhibition of
this process. In earlier studies, we demonstrated that the pathway of
monocyte CD40 signaling resulting in activation of inflammatory
cytokine synthesis and rescue from apoptosis is critically dependent on
the generation of PTK activity and does not show dependence on the
activity of the serine/threonine protein kinase C family (15, 16). IL-4
and IL-10 inhibited CD40-induced tyrosine phosphorylation of monocyte
cellular proteins, and down-regulated CD40-induced IL-1
in a
synergistic manner (16). In the present study we have evaluated the
role of MAPK family members in the CD40 signaling of inflammatory
cytokine production and the influence of IL-4 and IL-10 on MAPK
activity. CD40 signaling in monocytes resulted in the rapid
phosphorylation and accompanying activation of ERK1/2, whereas
phosphorylation of MAPK family members p38 and c-Jun N-terminal kinase
(JNK), was not observed. The data herein provide evidence that
CD40-mediated induction of IL-1
and TNF
synthesis is dependent on
MEK1/2 activity and demonstrate that activation of the MEK1/2/ERK1/2
pathway is a target for the inhibitory action of IL-4 and IL-10 in monocytes.
 |
MATERIALS AND METHODS |
Control of Endotoxin Contamination--
All cell culture
reagents used were either certified as low endotoxin when purchased, or
were ensured low endotoxin as determined by chromogenic limulus assay
(BioWhittaker, Walkersville, MD). Stock solutions containing >1 ng/ml
(10 endotoxin units/ml) were considered unacceptable. Stock solutions
were diluted in assays such that endotoxin levels did not exceed 1 pg/ml.
Reagents, Antibodies, and Cell Lines--
Sodium orthovanadate
(Na3VO4) was acquired from Fisher Scientific,
(Pittsburgh, PA). IL-4 and IL-10 were purchased from R&D Systems
(Minneapolis, MN). The MEK1/2 inhibitor PD98059 was obtained from New
England BioLabs, Inc. (Beverly, MA). The following mAbs were prepared
from culture supernatants of hybridomas purchased from the American
Type Culture Collection (ATCC, Rockville, MD): IgG mouse anti-human
IL-1
(H-6A), IgG mouse anti-human CD3 (OKT-3), IgG mouse anti-human
CD8 (OKT-8), IgG mouse anti-human monocyte (3C10), IgG mouse anti-human
B cell (LYM-1), and IgM mouse anti-human NK cell (hNK-1).
BioMagTM iron-conjugated goat anti-human IgG and IgM were
obtained from PerSeptive Diagnostics, Inc. (Cambridge, MA). IgG mouse
anti-human CD154 mAb was obtained from Genzyme (Cambridge, MA) and an
IgG isotype control mAb was purchased from Pharmingen (San Diego, CA).
Rabbit antibodies recognizing the active, phosphorylated (Thr183 and Tyr185) form of ERK1/2 were
acquired from Promega (Madison, WI) and from New England BioLabs.
Rabbit antibodies recognizing phosphorylated p38 (Thr180
and Tyr182) and phosphorylated JNK (Thr183 and
Tyr185) were purchased from New England BioLabs.
Horseradish peroxidase-conjugated F(ab')2 donkey
anti-rabbit Ab was purchased from Jackson ImmunoResearch Laboratories
(West Grove, PA).
Cell lines used in these studies included 293 (human embryonic kidney,
ATTC), stable transfectants of 293 which express high levels of CD154
(36), the human T cell leukemia line Jurkat (ATTC), and the
CD154+ sublcone of Jurkat, D1.1 (37). Both D1.1 and the
293-CD154 transfectants were gifts of Dr. Seth Lederman, Columbia
University. Cell lines were maintained in RPMI 1640 (Hyclone, Logan,
UT), containing 100 mM HEPES, 50 µg/ml gentamicin, and
fetal bovine serum at 5% (henceforth designated as R-5). The 293-CD154
transfectants (created by co-transfection with pcDNA1-CD154 and
pRSVneo) were periodically passaged in 200 µg/ml G418 (Life
Technologies, Inc.).
Monocyte Isolation and Culture--
Blood was collected from
normal, healthy human volunteers and peripheral blood mononuclear cells
were isolated over a Ficoll density gradient (Fico-Lite-LymphoH,
Atlanta Biologicals, Norcross, GA). Peripheral blood mononuclear cells
were plated at a density of 5 × 106 cells/well in
24-well tissue culture plates or at 5 × 105/well in
96-well plates (Falcon Primaria, Lincoln Park, NJ) in R5. Monocytes
were isolated by plastic adherence for 1 h at 37 °C after which
nonadherent cells were removed by Pasteur pipetting during 2 washes
with Dulbecco's phosphate-buffered saline. For use in immune complex
kinase assays, monocytes were purified from peripheral blood
mononuclear cells by counterflow elutriation as described previously
(38).
CD4+ T Cell Purification and
Activation--
CD4+ T cells were purified by negative
magnetic panning from elutriation-enriched T cell populations. Cells
were incubated in R-5 with mAbs against cell surface molecules
generated from the hybridomas OKT-8 (anti-CD8+ T cell),
3C10 (anti-monocyte), LYM-1 (anti-B cell), and hNK-1 (anti-NK cell),
used as culture supernatants at dilutions of 1:10, for 30 min at room
temperature. Cells were then treated with BioMagTM
iron-conjugated antibodies to murine IgG and IgM (PerSeptive Diagnostics, Cambridge, MA) for 30 min with gentle shaking at 4 °C.
Cells were diluted with Dulbecco's phosphate-buffered saline in
75-cm2 flasks (Fisher Scientific) and the CD4
populations were removed via 27 megagauss Oerstead magnets (PerSeptive Diagnostics). A sample of the purified population was stained with an
fluorescein isothiocyanate-conjugated anti-CD4 mAb and analyzed by flow
cytometry on a FACSTARTM Plus flow cytometer (Becton
Dickinson, San Jose, CA). Resulting populations were typically found to
be greater than 95% CD4+. CD4+ T cells were
then rested in R-5 alone, or activated for 6 h in R-5 by
incubation with 10 ng/ml phorbol 12-myristate 13-acetate (Sigma) and
0.5 µM ionomycin (Calbiochem, San Diego, CA). Expression of CD154 on activated, but not resting, CD4+ T cells was
then confirmed by flow cytometric analysis.
T Cell Plasma Membrane Preparation--
For the preparation of
purified plasma membranes, resting and activated purified
CD4+ T cells were resuspended in a hypotonic buffer
containing 50 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM MgCl2, and 50 µg/ml phenylmethylsulfonyl fluoride for 30 min on ice. The cells were then homogenized using a
PowerGen 35 homogenizer (Fisher Scientific) until completely disrupted
as determined microscopically. Disrupted cells were centrifuged at
500 × g for 5 min to remove nuclei, then centrifuged at 95,000 × g for 30 min using a Ti-50 rotor in a
Beckman L5-65 Ultracentrifuge. Cell debris was resuspended in 35%
(w/v) sucrose/hypotonic buffer then layered on 73% (w/v)
sucrose/hypotonic buffer. Hypotonic buffer was layered on the 35%
sucrose and the samples were centrifuged using a SW50.1 rotor at
130,000 × g for 1 h to separate plasma membranes.
The plasma membrane layer (at the 73-35% interface) was collected and
diluted 1:5 with hypotonic buffer, then centrifuged again for 1 h
at 130,000 × g to pellet purified plasma membranes. The membrane pellets were resuspended in phosphate-buffered saline and
total protein was determined by microtiter plate protocol of the
bicinchoninic acid protein assay (Pierce, Rockford, IL). The
bicinchoninic acid protein assay was read on a Biotek Instruments microtiter plate reader at 561 nm.
Analysis of IL-1
and TNF
Synthesis--
Induction of
IL-1
production by monocytes was measured by metabolically labeling
the cells with 50 µCi/ml Tran35S-LabelTM (ICN
Radiochemicals, Irvine, CA) in methionine-deficient RPMI 1640 (Hyclone,
Logan, UT). After labeling, cells were rinsed with Dulbecco's
phosphate-buffered saline and lysed in cold immunoprecipitation buffer
containing 25 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1% deoxycholate, 0.35 M NaCl, 10 mM EDTA, and 50 µg/ml phenylmethylsulfonyl fluoride. IL-1
was immunoprecipitated
from the cell lysates with mouse anti-human IL-1
followed by
isolation with Immobilized rProtein-ATM (Repligen Corp.,
Cambridge, MA). Precipitates were analyzed by SDS-PAGE in 15% minigels
followed by autoradiography. For analysis of TNF
production
stimulated through CD40, monocytes were plated in 96-well plates in R5
and co-incubated with 293, 293-CD154 transfectants, Jurkat, or D1.1
cells. Supernatants were harvested after an 18-h incubation and assayed
by enzyme-linked immunosorbant assay using R&D system's
QuantikineTM TNF
enzyme-linked immunosorbant assay system.
Western Blot Analysis of MAPK Phosphorylation--
Prior to
stimulation, monocytes were pretreated with 100 µM
Na3VO4 for 20 min to negate protein tyrosine
phosphatase effects on tyrosine-phosphorylated cellular proteins during
stimulation. After monocyte treatment/stimulation in 24-well plates,
cells were lysed in 50 µl of boiling treatment buffer (125 mM Tris, pH 6.8, 2% SDS, 20% glycerol, 1%
-mercaptoethanol, and 0.003% bromphenol blue) containing 200 µM phenylmethylsulfonyl fluoride and 1 mM
sodium orthovanadate. Samples were resolved by SDS-PAGE in 15%
minigels. Proteins were transferred to BioBlot-NC nitrocellulose membranes (Corning Costar Corp., Kennebunk, ME) using a
Trans-BlotTM SD Semi-Dry Electrophoretic Transfer Cell
(Bio-Rad). Antibody-bound proteins were detected using an enhanced
chemiluminescence ECLTM Western blotting analysis system
(Amersham Corp.) and the membranes were exposed to Kodak X-Omat LS
x-ray film.
Immune Complex Kinase Assay--
Elutriation purified monocytes
were plated in 6-well plates at 3 × 106 well. After a
30-min stimulation period the cells were harvested in a lysis buffer
containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM
-glycerol
phosphate, 1 mM Na3VO4, 20 µg/ml
phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin. Active ERK1/2
was immunoprecipitated from the cell lysates with polyclonal rabbit
anti-phospho-ERK1/2 followed by incubation with Immobilized
rProtein-ATM. Immune complexes were suspended in a kinase
buffer containing 25 mM Tris, pH 7.5, 5 mM
glycerol phosphate, 2 mM dithiothreitol, 0.1 mM
Na3VO4, 10 mM MgCl2,
and 200 µM ATP to which 2 µg of Elk1 fusion protein
(New England BioLabs) was added for a 30-min incubation. The samples
were analyzed by SDS-PAGE and Western blot (as described above) using
phospho-specific Elk1 antibody (New England BioLabs) as a probe,
followed by horseradish peroxidase-conjugated F(ab')2 goat
anti-rabbit antibody (Jackson ImmunoResearch Laboratories) for
detection using the ECLTM Western blotting analysis system.
X-ray films were analyzed by scanning densitometry using the
UN-SCAN-IT-gel automated digitizing system, Silk Scientific Corp.,
Orem, UT.
 |
RESULTS |
T Cell Activation of Monocytes through CD40 Results in Activation
of the ERK1/2 MAPKs--
Experiments were designed to evaluate the
role of MAPKs in T cell activation of monocytes through CD40 signaling.
Stimulus was provided by plasma membranes purified from
CD4+ T cells which had been activated for 6 h (a time
point at which CD154 expression was optimal) as well as by co-culture
of monocytes with CD154 transfectants, or the CD154+ Jurkat
T cell variant, D1.1 (36, 37). Controls included use of purified plasma
membranes from resting (CD154
) CD4+ T cells,
and the CD154
293 and Jurkat parent cell lines. Plasma
membrane preparations were titrated for activity based on membrane
protein concentrations. In the experiments presented herein membranes
were used at 10 µg of protein/ml. In these, and in previous
experiments (5, 15, 16), we have demonstrated that the activation of
monocytes induced through co-incubation with plasma membranes purified
from 6-h activated CD4+ T cells is inhibited by addition of
anti-CD154 antibodies (as is activation by CD154 transfectants and the
D1.1 cell line), indicating that the primary activating component of
this reagent is the CD40 ligand, CD154.
To evaluate the ability of CD40 stimulation to activate MAPKs,
monocytes were treated with plasma membranes purified from either
activated (CD154+) or resting (CD154
)
CD4+ T cells, designated TmA and TmR, respectively. After a
30-min incubation period, cell lysates were harvested and analyzed for MAPK activation by Western blot using antibodies specific for the
phosphorylated (active) forms of ERK1/2, p38, and JNK. In the first set
of experiments the influence of CD40 signaling on ERK1/2
phosphorylation was evaluated. Treatment of monocytes with TmA, but not
TmR (each at 10 µg/ml), resulted in the phosphorylation of ERK1 (44 kDa) and ERK2 (42 kDa) and pretreatment of TmA with anti-CD154 mAb, but
not with an isotype-matched control mAb, greatly reduced
CD40-dependent ERK1/2 activation (Fig.
1A). Although the samples
evaluated represent lysates generated from an equal number of cells,
the blot was stripped and re-probed with anti-
-actin as a loading
control ensuring that discrepancies in gel loading or electroblotting
had not occurred.
-Actin levels were equivalent between groups (Fig.
1A, lower panel).

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Fig. 1.
CD40 signaling results in activation of
ERK1/2. A, top panel, monocytes were left as
unstimulated (control) or treated for 30 min with TmR, TmA,
or TmA + antibody, as indicated. Cell lysates were analyzed on SDS-PAGE
followed by Western blotting using a polyclonal antibody directed
against the dually phosphorylated ERK1 (44 kDa) ERK2 (42 kDa).
Bottom panel, the Western blot (above) was stripped and
re-probed with anti- -actin mAb as a loading control. B,
immune complex kinase assay of CD40-mediated ERK activation. Monocytes
were either left as unstimulated (control) or stimulated
with TmA as indicated. After a 30-min incubation, monocyte lysates were
immunoprecipitated with anti-phospho-ERK1/2 antibody. The pelleted
immunoprecipitates were incubated with an Elk1-glutathione
S-transferase fusion protein as a substrate and
phosphorylation of Elk1 was visualized by Western blot using antibody
specific for phosphorylated Elk1. Panels A and B
are representative of two separate experiments which yielded similar
results.
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|
The induction of ERK1/2 phosphorylation would be expected to be
accompanied by enhanced kinase activity. The induction of ERK1/2 kinase
activity via CD40 signaling was assessed using an immune complex kinase
assay. ERK1/2 was immunoprecipitated from control and TmA-treated
monocyte lysates using anti-phospho-ERK1/2 antibodies. The resulting
immunoprecipitates were incubated with an Elk1-GST fusion protein and
phosphorylation of Elk1 was assayed by Western blot using a
phospho-specific Elk1 antibody. Induction of ERK1/2 kinase activity
through CD40 stimulation was confirmed by the phosphorylation of Elk1
substrate apparent in the TmA, but not in the control monocyte lysates
(Fig. 1B).
The ability of TmA to induce MAPK phosphorylation was evaluated over a
2-h time period postactivation. ERK1/2 phosphorylation was evident at
10 min, and declined at 2 h (Fig.
2A, top panel). Probing the
blot with antibody recognizing both phosphorylated and
nonphosphorylated forms of ERK present revealed that the differences observed were not due to differences in the level of ERK1/2 protein, or
artifacts of gel loading (Fig. 2, bottom panel). Although
LPS stimulation resulted in the phosphorylation of both p38 and JNK (p46), as previously reported (39, 40), treatment of monocytes with TmA
over the same 2-h time period examined in Fig. 2A, did not
induce or enhance p38 or JNK phosphorylation (Fig. 2B).
Probing with anti-phospho-p38 revealed a low level of phosphorylated
protein present in unstimulated monocytes. LPS enhanced the level of
phosphorylation of p38, whereas TmA stimulation had no influence of p38
phosphorylation at the time points tested. Phospho-JNK was undetectable
in control or TmA-stimulated cells. Lysates of anisomycin-treated
glioma cells and UV-treated 293 cells (New England BioLabs) were used as positive controls for anti-phospho-p38 and anti-phospho-JNK reactive
proteins, respectively (not shown).

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Fig. 2.
Analysis of MAPK phosphorylation in response
to CD40 signaling. A, analysis of ERK1/2
phosphorylation. Monocytes were either left unstimulated, or stimulated
with LPS for 30 min, or TmA for 10, 30, 60, and 120 min as indicated.
After stimulation, the lysates were harvested and analyzed by Western
blot with antibodies recognizing the phosphorylated form of ERK1/2
(top panel) or antibodies recognizing total ERK1/2
(bottom panel). B, analysis of p38 and JNK
phosphorylation. Monocytes stimulated as in A, above, were
lysed and the lysates analyzed by Western blot with antibodies
recognizing the phosphorylated forms of p38 and JNK, as indicated. The
data shown in panels A and B are representative
of two separate experiments.
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|
MEK Activity Is Required for CD40 Signaling of IL-1
and TNF
Synthesis in Monocytes--
We next addressed the question as to
whether the activation of ERK1/2 has functional significance in terms
of induction of inflammatory cytokine synthesis. The role of
CD40-mediated activation of ERK1/2 in the induction of inflammatory
cytokine production was explored via upstream blockade of the ERK1/2
pathway. Activation of ERK1/2 is catalyzed by the dual specificity
kinase MEK1/2, which, itself, is activated through serine
phosphorylation catalyzed by Raf family kinases (41). Use of the
specific MEK1/2 inhibitor, PD98059, which prevents Raf-mediated
activation of MEK1/2 (42), allowed us to evaluate the role of the
MEK/ERK pathway in CD40-mediated induction of IL-1
and TNF
synthesis. PD98059 effectively inhibited ERK1/2 phosphorylation in
monocytes as assayed by Western blot and had no adverse effects on cell
viability in a concentration range of 1-100 µM tested,
even during prolonged periods of treatment, as confirmed by examining
total de novo protein synthesis after metabolic labeling of
cells with [35S]methionine (not shown). To determine the
role of MEK activity in CD40 induction of IL-1
synthesis monocytes
were pretreated with PD98059 for 1 h prior to CD40 stimulation
with TmA. IL-1
induction was analyzed by radiolabeling of monocytes
during stimulus and immunoprecipitation of radiolabeled protein with
monoclonal anti-IL-1
antibody. Pretreatment of monocytes with
PD98059 effectively blocked CD40-mediated IL-1
synthesis induced by
TmA in a dose-dependent manner, as assayed by
immunoprecipitation of radiolabeled protein synthesized over a 4-h
period of stimulation with TmA (Fig.
3A).

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Fig. 3.
CD40 signaling of IL-1
and TNF synthesis in monocytes requires
MEK1/2 activity. A, CD40-mediated IL-1
synthesis in the presence of PD98059. Monocytes were preincubated for
1 h in the presence or absence of PD98059, then stimulated with
TmA for 4 h in the presence of [35S]methionine for
metabolic labeling of proteins. Cell lysates were harvested and
immunoprecipitated with monoclonal anti-IL-1 antibody. Lane
1, lysates from unstimulated monocytes; lane 2, TmA
stimulated monocytes; lane 3, TmA + 1 µM
PD98059; lane 4, TmA + 10 µM PD98059;
lane 5, TmA + 30 µM PD98059. The data shown is
representative of two separate experiments. B, CD40-mediated
TNF synthesis in the presence of PD98059. Monocytes were plated in
96-well tissue culture plates to which 293 control cells or 293-CD154
transfectants were added at 104 cell/well. 293-CD154 cells
were either incubated with untreated monocytes or with monocytes
pretreated with PD98059 at 5, 10, 30, or 60 µM as
indicated. After an 18-h incubation the cell supernatants were assayed
by TNF enzyme-linked immunosorbant assay. Two independent
experiments are displayed which are representative of four separate
experiments yielding similar results. C, as a specificity
control, monocytes were incubated with 293-CD154 or 293-CD154 + 1 µg/ml anti-CD154 as shown. The data in B and C
are presented as the mean of triplicate determinations of supernatant
TNF content + S.D.
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The influence of PD98059 on monocyte TNF
synthesis in response to
CD40 stimulation was analyzed by enzyme-linked immunosorbant assay of
monocyte supernatants. Monocytes were stimulated by addition of
CD154-transfected 293 cells (293-CD154) in the presence or absence of
PD98059, and supernatants were collected for analysis at 18 h.
Controls consisted of supernatants from monocytes stimulated with the
293 parent line. Monocyte TNF
synthesis was induced by interaction
with the CD154 transfectants, but not by co-culture with the control
293 population, and was suppressed by addition of PD98059 in a
dose-dependent manner (Fig. 3B). Similar results were obtained using TmA or the CD154+ D1.1 Jurkat subclone as stimulus.
In both cases PD98059 inhibited TNF
induction in a similar fashion
to that shown in Fig. 3B. The level of blockade of TNF
synthesis varied dependent on the overall level of response, with a
robust response requiring higher levels of the compound for inhibition
than experiments in which a lower overall response was observed (due to
donor variation), as shown in the two experiments selected for display
(Fig. 3B). Complete inhibition of CD40-induced TNF
synthesis was observed at 60 µM, a concentration that
effectively inhibits activation of both MEK1 and MEK2 (42). The
specificity of the role of CD40-CD154 interactions in the induction of
TNF
in these experiments was confirmed by the ability of anti-CD154 to abrogate monocyte responses to this stimuli (Fig. 3C).
Isotype control antibodies had no effect on CD40-mediated induction of TNF
synthesis (not shown). These data indicate that MEK activation of ERK1/2 is a critical element of the CD40-mediated signaling pathway
leading to IL-1
and TNF
synthesis in monocytes.
IL-4 and IL-10 Inhibit CD40-mediated Activation of the MEK/ERK
Pathway--
In previous work IL-4 and IL-10 were found to inhibit
CD40-induced activation of PTK activity and the subsequent synthesis of
inflammatory cytokines (16). Given the results above indicating a role
of MEK and ERK activation in this pathway, we asked if IL-4 and IL-10
acted by interference of the signaling cascade leading to ERK1/2
activation. To address this question, monocytes were pretreated for an
18-h period with IL-4 or IL-10 prior to TmA stimulation and the cell
lysates were assayed for phosphorylation of ERK1/2. ERK1/2 activation
was decreased in a dose-dependent manner with both IL-4 and
IL-10 (Fig. 4). Quantification by
scanning densitometry indicated that a dose of 50 ng/ml IL-4 resulted
in 79% decrease in the level of phosphorylation observed over
background in response to TmA. A 90% decrease was observed with
treatment of IL-10 at 50 ng/ml. Neither of these cytokines affected the level of expression of ERK's during this time period as shown by
Western blots of the same samples, re-probed with an antibody reactive
with total ERK1/2, which show equivalent levels of ERK1/2 regardless of
treatment (Fig. 4, bottom panel). In our earlier work, we
demonstrated that IL-4 and IL-10 could act synergistically in the
down-regulation of IL-1
synthesis and PTK activity (16). Likewise,
activation of ERK1/2 was reduced further with a combination of IL-4 and
IL-10 than with either cytokine alone (Fig.
5, lane 5). In the experiment
shown, IL-4 and IL-10 at 10 ng/ml reduced TmA-induced ERK activation by
approximately 50%, based on scanning densitometry (Fig. 5,
middle panel), whereas the combination of IL-4 and IL-10 at
5 ng/ml each reduced phosphorylation of ERK1/2 resulted in a 90%
reduction in the level of phosphorylation seen in response to TmA (Fig.
5, lane 5). Re-probing with anti-
-actin as a loading
control ensured that the differences observed were not due to artifacts
of gel loading or electroblotting (Fig. 5, bottom
panel).

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Fig. 4.
Preincubation of monocytes with IL-4 and
IL-10 inhibits ERK1/2 activation and does not affect ERK
expression. Top panel, monocytes preincubated with IL-4
and IL-10 at 5, 10, and 50 ng/ml, as indicated, were analyzed by
Western blot for level of ERK1/2 phosphorylation using
anti-phospho-ERK1/2 antibodies. Middle panel, the film shown
in the top panel was scanned and the digitized image
analyzed for band density. The histogram represents density (total
pixels minus background × 10 3) of the ERK2 bands.
Bottom panel, the Western blot probed with
anti-phospho-ERK1/2 (top panel) was stripped and re-probed
with an anti-ERK1/2 which recognizes both active and inactive forms of
the kinase.
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Fig. 5.
IL-4 and IL-10 inhibit CD40-mediated ERK1/2
phosphorylation. Top panel, monocyte lysates were
analyzed by Western blot for level of ERK1/2 phosphorylation using
anti-phospho-ERK1/2 antibodies. Lane 1, TmR treated;
lane 2, TmA treated; lane 3, TmA + 10 ng/ml IL-4;
lane 4, TmA + 10 ng/ml IL-10; lane 5, TmA + IL-4
and IL-10 at 5 ng/ml each. The data shown are representative of four
separate experiments. Middle panel, the film shown
(top panel) was scanned and the digitized image analyzed for
band density. The histogram represents density (total pixels minus
background × 10 3) of the ERK2 bands. Bottom
panel, the Western blot probed with anti-phospho-ERK1/2 (top
panel) was stripped and re-probed with anti- -actin.
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IL-4 and IL-10 inhibition of ERK1/2 kinase activity was demonstrated by
assay of ERK1/2 kinase activity in anti-phospho-ERK1/2 immunoprecipitates of monocytes treated with TmA in the presence of
IL-4 and IL-10. Monocytes were stimulated with TmA as above in the
presence of IL-4 or IL-10 individually at 10 ng/ml, or the combination
of the two cytokines at 5 ng/ml each. IL-4 and IL-10 inhibited ERK1/2
kinase activity as measured by phosphorylation of the Elk1 substrate
when used independently, and displayed synergy when combined (Fig.
6A). Scanning densitometry
indicated that IL-4 reduced ERK1/2 kinase activity as measured by
phosphorylation of Elk1 by approximately 70% of that induced by TmA,
whereas a 90% reduction in kinase activity was observed with IL-10
treatment (Fig. 6, lanes 4 and 5, respectively).
The combination of IL-4 and IL-10 reduced ERK1/2 kinase activity to the
background value (Fig. 6, lane 6).

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Fig. 6.
IL-4 and IL-10 inhibit CD40-mediated
activation of ERK1/2 kinase activity. A, monocytes were
either left untreated or preincubated 18 h with IL-4, IL-10, or a
combination of the cytokines. The cells were then left untreated or
treated with TmR or TmA. Lane 1, unstimulated; lane
2, TmA treated; lane 3, TmR treated; lane 4,
TmA + 10 ng/ml IL-4; lane 5, TmA + 10 ng/ml IL-10;
lane 6, TmA + IL-4 and IL-10 at 5 ng/ml each. After a 30-min
incubation the lysates were immunoprecipitated with anti-phospho-ERK1/2
and ERK1/2 kinase activity was assessed as in Fig. 1B, using
an Elk1 fusion protein substrate. B, the film shown in
panel A was scanned and the digitized image analyzed for
band density. The histogram represents density (total pixels minus
background × 10 3) of the bands reactive with
anti-phospho-Elk1 antibody.
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DISCUSSION |
Over the past several years data has accumulated demonstrating
that CD40-CD154 interactions contribute to normal cell-mediated responses as well as to chronic inflammatory disease (12). In the case
of autoimmune inflammatory disease, studies thus far suggest a scenario
whereby self-reactive CD154+ T cells activate resting
CD40+ monocytes/macrophages resulting in production of
inflammatory mediators and prolongation of monocyte lifespan, with the
net effect being the maintenance or aggravation of the inflammatory process. The signaling pathways activated through CD40 in monocytes have not been clearly delineated. However, CD40 signaling in B cells
has been reported to involve activation of src family PTKs, serine/threonine kinases, Jak3, MAPK family members (JNK, p38, and
ERK1/2), and phospholipase-
2 (43-47). Downstream events in CD40-mediated signaling in B cells include the activation of
transcription factors NF
B (48, 49), NFAT, AP-1 (50), and STAT6 (51). As is the case for other members of TNF receptor superfamily, CD40,
itself, does not contain cytoplasmic sequences with catalytic activity
and has been shown to employ adapter proteins of the TNF
receptor-associated factor family as a means to mediate intracellular signaling events (52-54). Signaling through CD40 as well as through TNF receptor stimulation has been shown to initiate TNF
receptor-associated factor-mediated activation of MAPK family members.
TNF receptor-associated factor 2 has been shown to mediate JNK
activation in response to TNF
(55) and expression of TNF
receptor-associated factor 6 was found to be associated with activation
of ERK2 in a co-transfection system using the 293 human embryonic
kidney cell line (56).
Thus far, studies dealing with CD40-mediated responses in monocytes
have established that induction of PTK activity is an early event in
this pathway and is required for both the activation of inflammatory
cytokine synthesis as well as rescue from apoptosis (15, 16). The
present study evaluated the potential role of MAPK family members as
downstream mediators of CD40 signaling in monocytes. ERK1/2, p38, and
JNKs were all considered as likely candidates as mediators of CD40
signaling in monocytes based on several criteria. For example, each of
the three MAPKs have been implicated in both LPS activation of
monocytes (40, 57), as well as in CD40-mediated activation of B cells,
as mentioned above (58-63). In addition, JNK isoforms and p38 have
been shown to mediate signaling by members of the TNF receptor
superfamily (64, 65), of which CD40 is a member. Studies of MAPK
activation in B cell models have yielded mixed results, which may be a
result of the diversity of the source of B cells used in these studies,
as well as the use of various transformed B cell lines. The work
presented herein employed primary human monocytes, exclusively, to
evaluate the role of MAPKs in monocyte responses to ligation of CD40
with CD154. The stimulus used in these studies, which included plasma membranes purified from activated (CD154+) CD4+
T cells and CD154 transfectants, allowed for cross-linking of CD40
through interaction with the physiological form of CD154, which exists
in the membrane as heteromultimeric complexes (66).
Our data demonstrate that CD40 signaling in monocytes results in rapid
phosphorylation and activation of ERK1/2 (Figs. 1 and 2), whereas over
the same time period of stimulation, phosphorylation of p38 and JNK was
not increased above background (Fig. 2). As expected, the CD40-mediated
phosphorylation of ERK1/2 was accompanied by enhanced kinase activity
as shown by immune complex kinase assay of lysates from CD40-stimulated
monocytes (Fig. 1B). Thus far, ERK1/2 is the only
characterized substrate of the MAP kinase kinase MEK1/2. Therefore, the
ability of the MEK1/2 inhibitor PD98059 to abrogate CD40-mediated
IL-1
and TNF
production (Fig. 3) ascribes functional significance
to ERK1/2 activation in the pathway leading to inflammatory cytokine
synthesis. The nature of ERK1/2's contribution to
initiation/enhancement of TNF
and IL-1
transcriptional control is
a matter of ongoing investigation. Both genes are regulated, in part,
by the action of nuclear factor
B (NF
B) (67, 68) and nuclear
factor IL-6 (69), in addition to other transcription factors, including
AP-1(70) and PU.1 (71). Of these, nuclear factor IL-6 is known to be a
direct substrate of ERK1/2 catalytic activity (72), whereas the
activity of AP-1 components are influenced by ERK1/2 through increased
expression of c-Fos as a downstream result of ERK1/2 phosphorylation of
Elk1, rather than via direct activation (73).
Although p38 and JNK do not appear to be phosphorylated in response to
CD40 stimulation over the 2-h time period evaluated, we have not ruled
out the possibility that these MAPKs may be involved in later signaling
events resulting from autocrine IL-1
and/or TNF
stimulation.
Although TmA stimulation did not enhance phosphorylation of p38 above
background levels during the 2-h time period examined, in some
experiments, addition of the compound SB203580, an inhibitor of p38
activity (74), reduced TNF
production in response to CD40
stimulation over an 18-24-h period (not shown). The possibility that
this reduction is due to an inhibition of IL-1
autocrine
stimulation, resulting in p38 activation and enhancement of TNF
production (75, 76), is being investigated.
A goal of this line of research is not only to gain an understanding of
the means by which CD40 signaling leads to inflammatory cytokine
synthesis in monocytes, but also to determine how this process can be
suppressed. To this end we have evaluated the ability of the
anti-inflammatory cytokines IL-4 and IL-10 to modulate CD40 signaling
in monocytes. In earlier studies we demonstrated that both IL-4 and
IL-10 inhibited CD40-induced PTK activity, and dramatically
down-regulated CD40-induced IL-1
production in a synergistic manner.
IL-4 and IL-10 did not significantly lower CD40 surface expression and
were effective when added at the same time as stimulus, indicating a
direct effect on the CD40 signaling pathway (16). The data herein show
that both cytokines reduced CD40-induced phosphorylation of ERK1/2 and,
in concordance with the ability of the two cytokines to synergize in
down-regulating cytokine production, the data presented in Fig. 5
suggest that they may be synergistic in their ability to reduce
activation of ERK1/2, as well, since 5 ng/ml of both cytokines reduced
ERK1/2 phosphorylation to a greater degree than 10 ng/ml of either
cytokine alone. The reduced phosphorylation of ERK1/2 in IL-4- and
IL-10-treated monocytes correlated with reduced kinase activity as
measured by immune complex kinase assay (Fig. 6).
IL-4 and IL-10 appear to act early in the CD40 signal transduction
pathway. They reduce overall PTK induction, which we have shown is a
requirement for cytokine synthesis by the ability of PTK inhibitors to
effectively block CD40-activated cytokine synthesis (16). Not
surprisingly, PTK inhibitors block CD40-mediated activation of ERK1/2
(not shown). Our results are similar to those obtained in a study of
IL-10 effects on LPS mediated signaling, in which IL-10 was shown to
block LPS activation of PTK activity and the subsequent activation of
Ras and Raf-1 (77). The involvement of ERK1/2 in CD40 signaling
suggests that CD40 also triggers activation of the classical Ras/Raf-1
pathway and this possibility is being explored.
Both IL-4 and IL-10 signal through receptors which associate with and
activate Janus family (Jak) tyrosine kinases upon ligation. Substrates
of Jak's include the STAT (signal transducers and activators of
transcription) family of transcription factors (78) and IL-10 has been
shown to activate STATs 1 and 3 (79) in monocytes/macrophages. STATs
are responsible for the induction of a family of negative regulators of
cytokine signaling termed SOCS (suppressor of cytokine signals) which
act either directly or indirectly to inhibit Jak activity as a negative
feedback mechanism (80). Although, a recent report presented evidence
that CD40 may associate with Jak3 in B cells (46), CD40-mediated
activation of Jaks in monocytes has not been investigated. If a
functional association of Jak kinases and CD40 exists in monocytes,
this would open the possibility of SOCS modulation of CD40 signaling.
However, as mentioned previously, IL-4 and IL-10 can implement
inhibition when added simultaneously with stimulus (16). Although the
inhibitory effect is less dramatic than when the cells are pretreated
with the cytokines, the data do indicate that inhibition can be
achieved through rapid responses to IL-4 and IL-10 signaling that are
less likely to require transcriptionally controlled events. The data
indicate that IL-4 and IL-10 essentially "undo" the early
PTK-initiated signals generated through CD40 ligation, suggesting that
these cytokines may initiate or enhance tyrosine phosphatase activity,
leading to suppression of CD40 signaling. The synergistic nature of
IL-4 and IL-10 inhibition of CD40 signaling implies that these
cytokines must employ divergent mechanisms to impede the MEK/ERK pathway.