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INTRODUCTION |
The generation of an immune response involves the activation of
effector cells such as macrophages, neutrophils, and T lymphocytes and
the subsequent production of cytokines, chemokines, and reactive oxygen
and nitrogen intermediates. The activated macrophages are widely
recognized as cells that play an important role in inflammatory processes, as well as in the initiation, maintenance, and control of
specific immune responses. In response to
LPS1 and other activating
agents, macrophages secrete nitric oxide and proinflammatory cytokines,
such as TNF
, IL-1
, IL-6, and IL-12, and immunomodulatory
cytokines, such as TGF1 and IL-10 (1). Because the intensity and
duration of an inflammatory process depends on the local balance
between pro- and anti-inflammatory factors, the so-called "macrophage
deactivating factors" received considerable attention lately
(2-6).
Vasoactive intestinal peptide (VIP) and the pituitary adenylate
cyclase-activating polypeptide (PACAP) are two neuropeptides that
perform a broad spectrum of biological functions affecting both natural
and acquired immunity (reviewed in Refs. 7-9), primarily as
anti-inflammatory agents. VIP and PACAP have been shown to inhibit T
cell proliferation and cytokine production (reviewed in Ref. 10) and to
inhibit several macrophage functions, including phagocytosis,
respiratory burst, and chemotaxis (reviewed in Ref. 8). In agreement
with their anti-inflammatory role, VIP and PACAP were recently reported
to inhibit the in vitro and in vivo production of
proinflammatory cytokines such as IL-6 and TNF (11-13), to reduce
the expression of the inducible nitric-oxide synthase (14), to enhance
the production of the anti-inflammatory cytokine IL-10 (15), to protect
mice from endotoxic shock presumably through the inhibition of
endogenous TNF and of other inflammatory mediators (16), and to act
as survival factors against tissue injury of lung and neuronal cells
(17-19). Furthermore, we and others have recently demonstrated that
VIP and PACAP inhibit IL-12 production in endotoxin-stimulated
peritoneal macrophages (20-22), with a subsequent inhibitory effect on
IFN
synthesis by T cells (22).
IL-12, an early proinflammatory cytokine secreted by macrophages
activated by microbial products, plays a central role in the regulation
of cell-mediated immunity (reviewed in Refs. 23-25). IL-12 stimulates
the proliferation of activated T lymphocytes and enhances IFN
secretion by NK cells and T lymphocytes. Consistent with this latter
effect, IL-12 has a pivotal role in the induction of CD4+
Th1 cell responses, acting in antagonism to IL-4, the major promoter of
the Th2 response (26, 27). In mice, IL-12 plays a decisive role in the
protection against intracellular pathogens, including parasites and
bacteria (23-25). Thus, the understanding of the mechanisms that
affect IL-12 production in normal and pathological conditions could
contribute to immune response-based therapies or vaccine designs.
IL-12 is a unique cytokine because of its heterodimer structure.
Bioactive IL-12 (p70) is composed of two disulfide-linked subunits (p35
and p40) encoded by two separate genes. When both subunits are produced
within the same cell, they assemble into a biologically active
heterodimer (28). However, although the expression of the p35 gene is
constitutive in a wide variety of cells, the p40 gene is highly
tissue-regulated, being restricted to phagocytic cells with
antigen-presenting capability (29, 30), and is therefore considered to
function as the regulatory component for IL-12 expression (29).
The key role of IL-12 in the immune response and in inflammation and
the importance of this cytokine in anti-tumor resistance have raised
considerable interest in the mechanisms of IL-12 gene transcription.
Two functional promoter regions of the p40 gene that confer LPS
inducibility and IFN augmentation have been identified (31-34). The
region spanning from position 
132 to 122 contains a novel NF-
B
site (31), whereas the region from 211 to 207 contains an Ets-2
element that binds a complex formed by the protein transactivators
Ets-2, GLp109, IRF-1, and c-Rel (32-34). The aim of this study was to
understand the molecular mechanisms through which VIP and PACAP inhibit
IL-12 production in macrophages stimulated with endotoxin and
IFN.
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EXPERIMENTAL PROCEDURES |
Reagents--
Synthetic VIP, PACAP38, VIP1-12, and
VIP10-28 were purchased from Calbiochem-Novabiochem (San
Diego, CA). The VPAC1 antagonist (Ac-His1,
D-Phe2, Lys15, Arg16,
Leu27) VIP (3-7)-GRF (8-27) and the VPAC1 agonist
(Lys15, Arg16, Leu27) VIP
(1-7)-GRF (8-27) were kindly donated by Dr. Patrick Robberecht (Université Libre de Bruxelles, Brussels, Belgium). The PAC1 antagonist PACAP6-38, secretin, and glucagon were obtained from Peninsula Laboratories (Belmont, CA). Oligonucleotides were synthesized by the Oligonucleotide Synthesis Service from Rutgers University (Newark, NJ). Murine recombinant mrIFN was purchased from
Pharmingen (San Diego, CA). LPS (from Escherichia coli
055:B5), calphostin C, forskolin, prostaglandin E2
(PGE2), protease inhibitors, phenylmethylsulfonyl fluoride,
EDTA, glycine, glycerol, EGTA, and dithiothreitol were purchased from
Sigma, and H89 was from ICN Pharmaceuticals Inc (Costa Mesa, CA).
Antibodies against IRF-1, Ets-2, CREB, and NF-B (p50, c-Rel, IB,
phosporylated IB, and p65) were obtained from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA).
Cell Cultures--
Mouse peritoneal macrophages were elicited by
intraperitoneal injection of 2 ml of 4% Brewer's thioglycollate
medium (Difco, Detroit, MI) into male Balb/c mice (age 6-10 weeks).
Peritoneal exudate cells were obtained 72 h after injection by
peritoneal lavage with ice-cold RPMI 1640 medium. Peritoneal exudate
cells containing lymphocytes and macrophages were washed twice and
resuspended in ice-cold RPMI 1640 medium supplemented with 2%
heat-inactivated fetal calf serum (Life Technologies, Inc.), containing
10 mM HEPES buffer, 1 mM pyruvate, 0.1 M nonessential amino acids, 2 mM glutamine, 50 mM 2-mercaptoethanol, 100 units/ml penicillin, and 10 µg/ml streptomycin (RPMI 1640 complete medium). Cells were seeded in flat bottom 96-well microtiter plates (Corning Glass, Corning, NY) at
8 × 104 cells/well in a final volume of 200 µl. The
cells were incubated at 37 °C for 2 h to allow adherence to
plastic, and nonadherent cells were removed by repeated washing with
RPMI 1640 medium. At least 96% of the adherent cells were macrophages
as judged by fluorescence-activated cell sorter analysis.
Raw 264.7 mouse macrophage cells (ATCC, Manasses, VA) were cultured in
Dulbecco's modified Eagle's medium supplemented with 2 mM
L-glutamine, 100 units/ml penicillin, 10 µg/ml
streptomycin, and 10% fetal calf serum (CM). The cells (8 × 104) were plated in flat bottom 96-well microtiter plates
in 200 µl of CM for 24 h. Nonadherent cells were removed by
aspiration and two washings with Dulbecco's modified Eagle's medium.
Macrophage monolayers (murine peritoneal macrophages or Raw 264.7 cells) were incubated in CM and stimulated with 200 units/ml IFN
before (8 or 12 h) or simultaneously (0 h) with treatment with LPS
(0.5 µg/ml). VIP or PACAP38 (10
12-107
M) were added at the same time with IFN or LPS. Control
cultures were treated with medium alone. Cell-free supernatants were
harvested at the designated time points and kept frozen (20 °C)
until IL-12 determination by ELISA.
Quantitation of IL-12 p40 and IL-12 p70
Production--
Secretion of IL-12 p40 and IL-12 p70 into culture
supernatants was quantitated by capture ELISAs as described previously
(22, 35). For the detection of IL-12 p40, the mAb C15.6 (Pharmingen) was used to coat the microtiter plates, followed by detection with the
biotinylated mAb C17.8 (Pharmingen). For the detection of IL-12 p70,
mAbs that recognize IL-12 p35 (Red-T/G297-289; Pharmingen) were used
to coat microtiter plates, followed by detection with the biotinylated
anti-IL-12 p40 mAb, C17.8. The sensitivity of ELISAs for IL-12 p40 and
IL-12 p70 were 20 and 15 pg/ml, respectively. The assay is specific for
IL-12, because other recombinant cytokines (IL-1, IL-2, IL-7, IL-6,
TNF, and IFN) do not bind above background levels.
RNA Extraction and Northern Blot Analysis--
Northern blot
analysis was performed according to standard methods. Macrophage
monolayers (2 × 106 cells/ml) were stimulated with
LPS (0.5 µg/ml) and IFN (200 units/ml), in the absence or presence
of VIP and PACAP (108 M) for different time
periods at 37 °C. Total RNA was extracted by the acid
guanidinium-phenol-chloroform method, electrophoresed on 1.2%
agarose-formaldehyde gels, transferred to Nytran membranes (Schleicher
& Schuell), and cross-linked to the nylon membrane using UV light.
Probes for IL-12 was synthesized by reverse transcription polymerase
chain reaction using primer pairs specific for the genes encoding IL-12
p35 and IL-12 p40 (36), as described previously (35). The 5' and 3'
primers used to generate the IRF-1 and IB probes were:
5'-TTGAACAGTCTGAGTGGCAGC-3' and 5'-ACTGACCCAAGGAGGATGGTC-3' for IRF-1
and 5'-CTGGACTCCATGAAAGACGAGG-3' and 5'-CGATGCCCAGGTAGCCATGGAT-3' for
IB, as described previously (37-39). Oligonucleotides were end-labeled with [-32P]ATP by using T4 polynucleotide
kinase. The RNA-containing membranes were prehybridized for 16 h
at 42 °C and then hybridized at 42 °C for 16 h with the
appropriate probes. The membranes were washed twice in 2× SSC
containing 0.1% SDS at room temperature (20 min each time), once at
37 °C for 20 min, and once in 0.1× SSC containing 0.1% SDS at
50 °C (20 min). The prehybridization and hybridization buffers were
purchased from 5 Prime 
3 Prime, Inc. (Boulder, CO). The membranes
were exposed to x-ray films (Eastman Kodak Co.) and analyzed by densitometry.
Electrophoretic Mobility Shift Assay--
Nuclear extracts were
prepared by the mini-extraction procedure of Schreiber et
al. (40) with slight modifications. Raw 264.7 cells were plated at
a density of 107 cells/well in 6-well plates, stimulated,
washed twice with ice-cold phosphate-buffered saline/0.1% bovine serum
albumin, and scraped off the dishes. The cell pellets were homogenized
with 0.4 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml
pepstatin, and 1 mM NaN3). After 15 min on ice,
Nonidet P-40 was added to a final concentration of 0.5%, the tubes
were gently vortexed for 15 s, and nuclei were sedimented and
separated from cytosol by centrifugation at 12,000 × g
for 40 s. Pelleted nuclei were washed once with 0.2 ml of ice-cold
buffer A, and the soluble nuclear proteins were released by adding 0.1 ml of buffer C (20 mM HEPES, pH 7.9, 0.4 M
NaCl, 1 mM EDTA, 1 mM EGTA, 25% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml
pepstatin, and 1 mM NaN3). After incubation for
30 min on ice, followed by centrifugation for 10 min at 14,000 rpm at
4 °C, the supernatants containing the nuclear proteins were
harvested, the protein concentration was determined by the Bradford
method, and aliquots were stored at 80 °C.
Oligonucleotides corresponding to the NF-B half site (134/110)
(31) and to the Ets-2 motif (292/196) (32, 33) of the IL-12 p40
promoter were synthesized and annealed. Aliquots of 50 ng of the
double-stranded oligonucleotides were end-labeled with
[-32P]ATP by using T4 polynucleotide kinase. For EMSAs
with macrophage nuclear extracts, 20,000-50,000 cpm of double-stranded
oligonucleotides, corresponding to approximately 0.5 ng, were used for
each reaction. The binding reaction mixtures (15 µl) contain 0.5-1
ng of DNA probe, 5 µg of nuclear extract, 2 µg of
poly(dI-dC)·poly(dI-dC), and binding buffer (50 mM NaCl,
0.2 mM EDTA, 0.5 mM dithiothreitol, 5%
glycerol, and 10 mM Tris-HCl, pH 7.5). The mixtures were
incubated on ice for 15 min before adding the probe, followed by
another 20-min incubation at room temperature. Samples were loaded onto 4% nondenaturing polyacrylamide gels and electrophoresed in TGE buffer
(50 mM Tris-HCl, pH 7.5, 0.38 M glycine, and 2 mM EDTA) at 100 V, followed by transfer to Whatman paper,
drying under vacuum at 80 °C, and autoradiography. In competition
and antibody supershift experiments, the nuclear extracts were
incubated for 15 min at room temperature with the specific antibody (1 µg) or competing cold oligonucleotide (50-fold excess) before the
addition of the labeled probe.
Western Blot--
Lysates, cytoplasmic fractions, or nuclear
extracts (see above) containing 20-30 µg of protein were subjected
to reducing SDS-polyacrylamide gel electrophoresis (12.5%). After
electrophoresis, the gel was electroblotted in Tris-glycine buffer
containing 40% methanol onto a nitrocellulose membrane (Trans-blot,
Bio-Rad). The membrane was blocked with TBS-T buffer (10 mM
Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 5%
milk powder for 1 h at room temperature and then incubated with
primary antibodies (rabbit anti-mouse IgG) against IB (1:250),
phosphorylated IB (1:500), NF-B p50 (1:1000), NF-B p65
(1:1000), or IRF-1 (1:500) in TBS-T containing 1% milk powder for
2 h at room temperature. The membrane was washed with TBS-T and
incubated with the secondary antibody (goat anti-rabbit IgG conjugated
to horseradish peroxidase) at 1:5000 dilution for 1 h at room
temperature. After washing three times in TBS-T for 5 min each and once
in TBS for 5 min, the membrane was drained briefly and subjected to the
enhanced chemiluminiscence detection system (ECL, Amersham Pharmacia
Biotech). The x-ray films were exposed for 5-20 min.
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RESULTS |
VIP and PACAP Inhibit LPS-induced IL-12 Production by IFN-primed
Macrophages--
To investigate the effects of VIP and PACAP on IL-12
production, peritoneal macrophages were stimulated with IFN and/or
LPS in the absence or presence of various doses of VIP or PACAP, and the amounts of IL-12 p40 and p70 released in the culture supernatants were assayed by ELISA. IFN alone did not induce significant IL-12 p40 and p70 production, whereas LPS alone stimulated a modest increase
over that of unstimulated macrophages (data not shown). Macrophages
treated simultaneously with IFN and LPS produced marginally more
IL-12 than those stimulated with either IFN or LPS alone (Fig.
1A). However, pretreatment
with IFN followed 12 h later by LPS stimulation resulted in a
dramatic increase in both IL-12 p40 and p70 production (Fig.
1A). VIP and PACAP inhibited IL-12 p40 and p70 release by
IFN/LPS-stimulated macrophages (Fig. 1A). The addition of
VIP or PACAP at the time of IFN priming resulted in a slightly
higher inhibitory effect than when neuropeptides were added at the same
time with LPS (data not shown).
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Fig. 1.
VIP and PACAP inhibit IL-12 p40 and p70
production in LPS/IFN-stimulated
macrophages. A, VIP and PACAP inhibit IL-12 p40 and p70
in peritoneal macrophages stimulated with IFN and/or LPS. Peritoneal
macrophages (4 × 105 cells/ml) were cultured in the
presence of IFN (200 units/ml) and LPS (0.5 µg/ml) (left
panels) or primed with IFN for 12 h followed by the
addition of LPS (right panels). VIP or PACAP
(108 M) were added together with IFN and
LPS (left panels) or at the time of IFN priming
(right panels). Control cultures were incubated with medium
alone. Supernatants were collected 24 h later, and IL-12 p40 and
p70 release was determined by ELISA. Each result is the mean ± S.D. of six experiments performed in duplicate. B and
C, VIP and PACAP inhibit IL-12 in IFN-primed Raw 264.7 cells. B, time course for the inhibitory effect of VIP/PACAP
on IL-12 p40 and p70 production. Raw 264.7 cells (4 × 105 cells/ml) were pretreated with IFN (200 units/ml)
for 12 h before LPS stimulation (0.5 µg/ml). VIP or PACAP
(108 M) was added at the time of IFN
priming. Control cultures were incubated with medium alone.
Supernatants collected at different times were assayed for IL-12 p40
and p70 production by ELISA. C, dose-response curve for the
inhibitory effect of VIP and PACAP on IL-12 p40 and p70 production. Raw
264.7 cells (4 × 105 cells/ml) were pretreated with
IFN (200 units/ml) for 12 h before LPS stimulation (0.5 µg/ml). Different concentrations of VIP or PACAP were added at the
time of IFN priming. Supernatants were collected 18 h after LPS
stimulation and IL-12 p40 and p70 release was determined by ELISA. For
A-C, cells cultured in the absence of IFN and LPS with
VIP or PACAP did not produce detectable levels of IL-12 p40 and p70
(<30 pg/ml). Each result is the mean ± S.D. of six experiments
performed in duplicate.
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The murine macrophage cell line Raw 264.7 primed with IFN and
stimulated with LPS shows a similar pattern of regulation. VIP and
PACAP inhibit IL-12 p40 and p70 in a dose- and
time-dependent manner (Fig. 1, B and
C). The dose-response curves were similar for VIP and PACAP,
showing maximal effects at 108 M (Fig. 1,
B and C).
VIP and PACAP Inhibit IL-12 Production at a Transcriptional
Level--
To determine whether VIP/PACAP affect IL-12 transcription,
Raw 264.7 cells were primed with IFN or treated simultaneously with
LPS and IFN in the presence or absence of 108
M VIP or PACAP for 4, 8, and 16 h, and total RNA was
prepared and subjected to IL-12 p40 and p35 Northern blot analysis.
IL-12 p40 mRNA is absent in unstimulated cells, marginally induced
upon simultaneous treatment with IFN and LPS, and induced strongly upon pretreatment with IFN followed by LPS stimulation (Fig. 2A). In contrast, IL-12 p35
mRNA was constitutively expressed in unstimulated macrophages and
was not affected by IFN priming and/or LPS stimulation (Fig.
2A). VIP and PACAP inhibited IFN/LPS-induced IL-12 p40
mRNA, without affecting IL-12 p35 mRNA expression (Fig. 2).
These results indicate that both neuropeptides down-regulate steady-state IL-12 p40 mRNA levels.
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Fig. 2.
VIP and PACAP inhibit IL-12 p40
transcription. Raw 264.7 cells (2 × 106
cells/ml) were cultured in the presence of IFN (200 units/ml) and
LPS (0.5 µg/ml) or primed with IFN followed by LPS stimulation
8 h later. VIP or PACAP (108 M) were
added at the time of IFN/LPS stimulation, of IFN priming, or of
LPS stimulation. Cells incubated with medium alone were used as basal
IL-12 p35 and p40 mRNA level controls. Total RNA was extracted, and
the expression of IL-12 p35, IL-12 p40 and -actin mRNA was
analyzed by Northern blot analysis at the indicated time points.
Results are expressed in densitometric units normalized for the
expression of -actin. One representative experiment of three is
shown.
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VIP and PACAP Inhibit NF-B Binding to the IL-12 p40
Promoter--
Activation and nuclear translocation of members of the
NF-B/c-Rel family constitutes the hallmark of macrophage stimulation by proinflammatory cytokines and bacterial products (41). Although the
IL-12 p40 promoter contains a complex array of transactivating binding
sites, NF-B is essential for maximal IL-12 p40 transcription after
IFN/LPS stimulation (31, 32). To investigate whether VIP/PACAP
affects NF-B binding, we used electrophoretic mobility shift assays.
Treatment of Raw 264.7 cells with IFN followed by LPS stimulation
led to NF-B binding, and VIP and PACAP inhibited the binding (Fig.
3A). The NF-B binding was
competed by an excess of unlabeled homologous oligonucleotide (NF-B)
but not of nonhomologous oligonucleotide (CRE) (Fig. 3A).
Antibody supershift experiments indicate that the NF-B-binding
complexes in IFN/LPS stimulated macrophages contain p50, p65, and
c-Rel (Fig. 3B). Because VIP and PACAP almost completely
blocked NF-B binding, to identify the NF-B-binding factors, we
used a 10-fold excess of nuclear extract in the VIP treated samples.
The NF-B binding complexes contain p50, p65, and c-Rel (Fig.
3B), suggesting that although VIP significantly reduces
NF-B binding, it does not change the composition of the NF-B
binding complexes.
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Fig. 3.
VIP and PACAP inhibit the binding of NF-B
to the IL-12 p40 promoter. A, Raw 264.7 cells (1 × 106 cells/ml) were primed with IFN (200 units/ml) for
8 h before LPS stimulation (0.5 µg/ml). VIP or PACAP
(108 M) were added at the time of IFN
priming. Control cultures were incubated with medium alone. Nuclear
extracts were prepared 6 h after LPS stimulation, and NF-B
binding was assessed by EMSA using a radiolabeled oligonucleotide
containing the murine B half-site of the IL-12 p40 promoter.
Specificity was conducted by the addition of 50-fold excess of
unlabeled homologous (NF-B) or nonhomologous (CRE) oligonucleotides
to the nuclear extracts (Comp). B, identification
of the proteins bound to the NF-B site by supershift analysis.
Nuclear extracts from the IFN/LPS-stimulated macrophages (5 µg)
and from IFN/LPS plus VIP-treated macrophages (a 10-fold excess, 50 µg) were incubated with 1 µg of polyclonal antibodies against p65,
p50, c-Rel, or CREB for 20 min before adding the oligonucleotide probe.
Similar results were observed in four independent experiments.
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VIP and PACAP Modulate the Composition of the Ets-2-binding
Complexes--
Recently, it has been established that the Ets-2
element situated at position 211/207 in the IL-12 p40 promoter is
required for optimal transcription of the IL-12 p40 gene in macrophages (32-34). This region interacts with the nuclear complex F1, induced in
monocytic cells primed with IFN and stimulated with LPS. The F1
complex is composed of Ets-2, the nuclear factor GLp109, and the
additional components IRF-1 and c-Rel (33). Stimulation of Raw 264.7 cells with IFN/LPS led to an increase in Ets-2 binding compared with
unstimulated cells, and treatment with VIP or PACAP did not
significantly affect the binding (Fig.
4A). The specificity of the
Ets-2-binding activity was confirmed with homologous (Ets-2) or
nonhomologous (CRE) oligonucleotides as competitors (Fig.
4A). Antibody supershift experiments were performed to
determine the composition of the Ets-2-binding complexes. In
IFN/LPS-stimulated cells, the majority of the complexes were
supershifted by anti-Ets-2, anti-IRF-1, and anti-c-Rel Abs but not by
anti-CREB Abs (an irrelevant Ab) (Fig. 4B). In contrast, in
IFN/LPS-stimulated cells treated with VIP and PACAP, the complex was
supershifted only by the anti-Ets-2 Ab, with no supershift with
anti-IRF-1, anti-c-Rel, or anti-CREB Abs (Fig. 4B). This
indicates that in VIP/PACAP-treated cells, the Ets-2-binding complexes
contain Ets-2 and minor amounts, if any, of IRF-1 and c-Rel.
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Fig. 4.
VIP and PACAP regulate the composition of the
Ets-2-binding complexes. A, Raw 264.7 cells (1 × 106 cells/ml) were primed with IFN (200 units/ml) for
8 h before LPS stimulation (0.5 µg/ml). VIP or PACAP
(108 M) were added at the time of IFN
priming. Control cultures were incubated with medium alone. Nuclear
extracts were prepared 6 h after LPS stimulation, and the Ets-2
binding activity was assessed by EMSA using a radiolabeled
oligonucleotide (292/196 bp) containing the murine Ets-2 motif of
the IL-12 p40 promoter. Specificity was conducted by the addition of
50-fold excess of unlabeled homologous (Ets-2) or nonhomologous (CRE)
oligonucleotides to nuclear extracts (Comp). B,
identification of the proteins bound to the Ets-2 site. Nuclear
extracts were preincubated with 1 µg of anti-Ets-2, anti-IRF-1,
anti-c-Rel, or anti-CREB antibodies for 20 min prior to the addition of
the radiolabeled probe. Similar results were observed in four
independent experiments.
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Involvement of VPAC1 and cAMP in the Effects of VIP on B and
Ets-2 Binding--
Next we investigated whether the inhibitory effect
of VIP/PACAP on the IL-12 production could be related to occupancy of
specific receptors. The immunological actions of VIP and PACAP are
exerted through a family of VIP/PACAP receptors that were recently
reclassified (42): VPAC1 and VPAC2, which exhibit similar affinities
for the two neuropeptides and activate primarily the adenylate cyclase system, and PAC1, which exhibits a 300-1000-fold higher affinity for
PACAP than for VIP and activates both the adenylate cyclase and
phospholipase C systems (reviewed in Ref. 42). Previously we showed
that the inhibition of IL-12 production in peritoneal macrophages by
VIP/PACAP is mediated primarily through VPAC1 and involves both
cAMP-dependent and -independent transduction pathways (22).
We reached similar conclusions regarding the inhibition of IL-12
production in Raw 264.7 cells. A VPAC1 agonist (43) inhibited IL-12
transcription and release from IFN/LPS-induced Raw 264.7 cells to a
similar degree as VIP/PACAP (Fig.
5A). Also, a specific VPAC1
antagonist (44) reversed the inhibitory effect of VIP/PACAP (Fig.
5A). In contrast, PACAP6-38, an antagonist specific for PAC1 and to a lesser degree for VPAC2 (45), did not
reverse the effects of VIP and PACAP (Fig. 5A). The role of second messengers was investigated by using calphostin C (a protein kinase C inhibitor), H89 (a cAMP-dependent protein kinase A
inhibitor), and forskolin and PGE2 (two strict
cAMP-inducing agents). Similar to peritoneal macrophages, forskolin and
PGE2 inhibited IL-12 p40 release in IFN/LPS-stimulated
Raw 264.7 cells, although they showed less of an effect at lower
concentrations (10 and 100 nM) as compared with VIP and
PACAP (Fig. 5B). In addition, the involvement of cAMP is
supported by the results obtained with the two protein kinase
inhibitors. The VIP/PACAP inhibition of IL-12 transcription and release
from IFN/LPS-stimulated Raw 264.7 cells was not affected by
calphostin C but was partially reversed by H89 (Fig.
5B).
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Fig. 5.
Involvement of VPAC1 and cAMP in the
VIP/PACAP inhibition of IL-12 production. A, Raw 264.7 cells (4 × 105 cells/ml for ELISA and 1 × 106 cells/ml for Northern blot) were primed with IFN
(200 units/ml) for 12 h (ELISA) or 8 h (Northern blot) before
LPS stimulation (0.5 µg/ml). VIP (108 M),
PACAP (108 M), or the VPAC1 agonist
(108 M) were added at the time of IFN
priming. In some experiments, the VPAC1 antagonist (106
M) or PACAP6-38 (106
M) were added together with VIP or PACAP (108
M). H89 (100 nM), or calphostin C (100 nM) were added to stimulated cultures containing VIP,
PACAP, or medium (control). Supernatants collected 16 h after LPS
stimulation were assayed for IL-12 p40 production by ELISA. Total RNA
was extracted 8 h after LPS stimulation, and the expression of
IL-12 p40 and -actin mRNA was analyzed by Northern blot
analysis. Results are expressed in densitometric units normalized for
the expression of -actin. The dotted lines represent
control values from cultures incubated with IFN plus LPS. Each
result is the mean ± S.D. of three experiments performed in
duplicate. B, VIP, forskolin, or PGE2 (at
different concentrations) were added at the time of IFN priming
(left panel). Different concentrations of H89 or calphostin
C were added together with VIP or PACAP (108
M) (right panel). Supernatants collected 16 h after LPS stimulation were assayed for IL-12 p40 production by ELISA.
The dotted lines represent control values from cultures
incubated with IFN plus LPS. Each result is the mean ± S.D. of
three experiments performed in duplicate. C, H89 or
calphostin C (100 nM) were added together with VIP or PACAP
(108 M). Total RNA was extracted 8 h after LPS stimulation and the expression of
IL-12 p40 and -actin mRNA was analyzed by Northern blot
analysis. Results are expressed in densitometric units normalized for
the expression of -actin. The dotted lines represent
control values from cultures incubated with IFN plus LPS. Each
result is the mean ± S.D. of three experiments performed in
duplicate.
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Because the inhibitory effect of VIP on IL-12 production is mediated
primarily through VPAC1 and cAMP represents at least one of the second
messengers involved, we determined the effect of the VPAC1 antagonist
and of the cAMP-dependent protein kinase A inhibitor H89 on
the changes induced by VIP in B and Ets-2-binding complexes. The
VPAC1 antagonist, but not PACAP6-38, reversed the
inhibitory activity of VIP on NF-B binding and on the changes in the
composition of the Ets-2-binding complexes (Fig.
6). This is in accordance with the fact
that the VPAC1 agonist showed a similar effect than that of VIP on
NF-B- and Ets-2-binding complexes (Fig. 6). In contrast, H89 had a
more limited effect. H89 did not reverse the inhibitory effect on
NF-B binding; however, H89 affected the composition of the
Ets-2-binding complexes, i.e. it reversed the VIP-induced
supershift pattern obtained with the anti-IRF-1 Ab but not with the
anti-c-Rel Ab (Fig. 6). These results suggest that both the inhibition
of NF-B and the change in the composition of the Ets-2-binding
complexes by VIP are mediated through VPAC1, but only the reduction in
IRF-1 in the Ets-2-binding complexes is entirely
cAMP-dependent. This conclusion is supported by the fact
that forskolin (a cAMP inducer) did not affect NF-B binding and
reduced the presence of IRF-1, but not c-Rel, in the Ets-2-binding
complexes (Fig. 6).
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Fig. 6.
Involvement of VPAC1 and cAMP in the
VIP/PACAP regulation of NF-B and Ets-2 binding. Raw 264.7 cells
(1 × 106 cells/ml) were primed with IFN (200 units/ml) for 8 h before LPS stimulation (0.5 µg/ml). VIP
(108 M), the VPAC1 agonist (108
M), or forskolin (106 M) was
added at the time of IFN priming. The VPAC1 antagonist
(106 M), PACAP6-38
(106 M), or H89 (100 nM) was
added to stimulated cultures containing VIP. After incubation, nuclear
extracts were prepared and incubated with the Ets-2 (left
and middle panels) or NF-B (right panel)
oligonucleotides and subjected to EMSA. Ets-2-binding
complex. Lane 1, no Ab. Left panel,
supershift with anti-IRF-1 Ab. Middle panel, supershift with
anti-c-Rel Ab. Ets-2 binding is expressed as mean ± S.D.
(n = 3) of arbitrary units from densitometric analysis
of EMSAs. NF-B binding. Right panel, no Ab.
Similar results were observed in three independent experiments.
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VIP and PACAP Inhibit IFN/LPS-dependent Reduction of
IB Levels and Nuclear Translocation of the p65 and c-Rel Subunits
of NF-B--
Nuclear translocation of NF-B is preceded by the
phosphorylation and proteolytic degradation of IB (41). To
determine whether the VIP/PACAP inhibition of NF-B binding was due
to an effect on IB phosphorylation/degradation, we examined the
levels of cytoplasmic IB by Western blot. Significant lower
levels of IB were observed in IFN/LPS-stimulated cells,
compared with unstimulated cells (Fig.
7A). VIP and PACAP completely
blocked the IFN/LPS-induced reduction in IB (Fig.
7A). Next, to investigate whether VIP and PACAP block
IFN/LPS-induced reduction in IB levels by increasing IB
mRNA expression and/or by inhibiting IB phosphorylation and
further degradation, we examined the levels of IB mRNA and
cytoplamic phosphorylated IB protein by Northern blot and Western
blot analysis, respectively, in IFN/LPS-stimulated macrophages in
the presence or absence of VIP or PACAP. As Fig. 7B shows,
neither VIP nor PACAP affected IB mRNA steady-state levels.
However, both neuropeptides significantly inhibited IFN/LPS-induced phosphorylation of IB protein (Fig. 7C).
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Fig. 7.
Effect of VIP and PACAP on
IB, p65, p50, c-Rel, IRF-1, and Ets-2
complexes. Raw 264.7 cells (1 × 106 cells/ml)
were primed with IFN (200 units/ml) for 8 h before LPS
stimulation (0.5 µg/ml). VIP or PACAP (108
M) was added at the time of IFN priming. Control
cultures were incubated with medium alone. Cytosolic and nuclear
proteins were extracted 4 h after LPS stimulation. A,
Western blot analysis was performed for IB in the cytoplasmic
fraction and for p50, p65, and c-Rel in the cytoplasmic as well as
nuclear extracts. B, total RNA was extracted 1 h after
LPS stimulation, and the expression of IB mRNA was analyzed
by Northern blot analysis. One representative experiment of three is
shown. Lower panel, results are expressed in densitometric
units normalized for the expression of -actin. Each result is the
mean ± S.D. of three experiments performed in duplicate.
C, cytosolic proteins were extracted 40 min after LPS
stimulation, and Western blot analysis was performed for specific
phosphorylated IB. One representative experiment of three is
shown. D, Western blot analysis for Ets-2 was performed on
nuclear extracts. E, total RNA was extracted 3 h after
LPS stimulation, and the expression of IRF-1 and -actin mRNA was
analyzed by Northern blot analysis. Western blot analysis was performed
for IRF-1 on nuclear extracts. One representative experiment of three
is shown.
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Because NF-B activation requires the nuclear translocation of p65 or
c-Rel, we measured the levels of p65 and c-Rel proteins in cytoplasm
and nucleus. As expected, upon IFN/LPS treatment, the levels of p65
and c-Rel declined in the cytoplasm and concurrently increased in the
nucleus (Fig. 7A). Treatment with VIP and PACAP abolished
the IFN/LPS-dependent changes in nuclear and cytoplasmic p65/c-Rel levels (Fig. 7A). These results indicate that VIP
and PACAP inhibit the IFN/LPS-induced nuclear translocation of p65 and c-Rel, which is consistent with the inhibition of reduction in
cytoplasmic IB levels. In contrast to p65, c-Rel and IB, p50
levels were not affected by IFN/LPS, with or without VIP/PACAP (Fig.
7A).
Ets-2 Protein Complexes Are Not Affected by VIP and PACAP--
To
investigate the effect of VIP and PACAP on the expression of the Ets-2
protein, nuclear extracts from unstimulated and IFN/LPS-stimulated
Raw 264.7 cells in the absence or presence of VIP or PACAP were
analyzed by Western blot. As described previously (33), a single band
of approximately 54 kDa was present in unstimulated cells, whereas
several additional bands, GLp58, GLp109, Lp119, and Lp165, were
detected in IFN/LPS-stimulated cells (Fig. 7D). Treatment
with VIP or PACAP did not significantly modify this protein pattern
(Fig. 7D). These results suggest that the two neuropeptides
do not inhibit the synthesis of the Ets-2 components.
VIP and PACAP Inhibit IRF-1 Expression--
Next we investigated
the effect of VIP and PACAP on the expression and synthesis of IRF-1,
another component of the Ets-2-binding complex. Northern blot analysis
indicated that although IRF-1 mRNA was not detectable in
unstimulated cells, it was strongly induced in the
IFN/LPS-stimulated Raw 264.7 cells (Fig. 7E). VIP and
PACAP significantly reduced the levels of specific IRF-1 mRNA (Fig.
7E). In addition, Western blot analysis of nuclear extracts
confirmed the inhibitory effect of VIP and PACAP on IRF-1 expression
(Fig. 7E, lower panel).
Involvement of VPAC1 and cAMP in the Effect of VIP on IB
Degradation, the Nuclear Translocation of p65 and c-Rel, and IRF-1
Expression--
To correlate the involvement of VPAC1 and cAMP with
changes in NF-B and Ets-2 complexes at the protein level, we
investigated the effect of the VPAC1 antagonist and H89 on the changes
induced by VIP/PACAP in B and Ets-2-binding complexes. The
VIP-induced decrease in nuclear p65, c-Rel and IRF-1 and the blockage
in reduction of cytoplasmic IB levels were completely reversed by
the VPAC1 antagonist (Fig.
8A). However, H89 reversed
only the inhibitory effect of VIP on IRF-1 expression (Fig. 8) without
affecting p65, c-Rel, and IB changes (Fig. 8A). In
contrast, calphostin C did not affect VIP effect on IRF-1 expression
nor on NF-B complex changes (Fig. 8A). None of the
treatments affected the nuclear p50 levels (Fig. 8A). These
results indicate that the VIP-induced inhibition of IB reduction,
nuclear translocation of p65 and c-Rel, and IRF-1 expression are
mediated through VPAC1, but only the inhibition of IRF-1 expression is
entirely cAMP-dependent. This is also supported by the fact
that forskolin did not affect neither nuclear levels of p65 and c-Rel
or the cytoplasmic IB levels but, similar to VIP/PACAP, inhibited
IRF-1 expression (Fig. 8).
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Fig. 8.
Involvement of VPAC1 and cAMP in the
VIP/PACAP regulation of NF-B and Ets-2 proteins. Raw 264.7 cells (1 × 106 cells/ml) were primed with IFN (200 units/ml) for 8 h before LPS stimulation (0.5 µg/ml). VIP
(108 M) or forskolin (1 µM) was
added at the time of IFN priming in the presence or absence of the
VPAC1 antagonist (106 M), H89 (100 nM), or calphostin C (100 nM). Nuclear and
cytoplasmic extracts obtained 4 h after LPS stimulation were
analyzed by Western blotting with antibodies against p50, p65, c-Rel,
and IRF-1 (nuclear extracts) or IB (cytosolic fraction). One
representative experiment of three is shown.
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DISCUSSION |
Macrophages are widely recognized as cells that play a central
role in the regulation of immune and inflammatory activities, as well
as tissue remodeling (1). In response to antigens such as LPS,
macrophages secrete proinflammatory cytokines and oxidants such as
TNF, IL-6, IL-1, IL-12, and nitric oxide (1). VIP and PACAP are
potent anti-inflammatory agents that down-regulate the activation of T
cells and macrophages (7-10). VIP and PACAP were shown to modulate the
macrophage secretion of proinflammatory mediators such as TNF, IL-6,
nitric oxide (11-14), and recently IL-12 (20-22). Here we extend
these studies to the molecular mechanisms involved in the inhibitory
effect of VIP/PACAP on IL-12 production. We focused on the regulation
of the 40-kDa IL-12 subunit that is induced by bacterial stimulation in
phagocytic cells. Production of IL-12 is maximal when macrophages are
primed with IFN for 12-18 h prior to LPS stimulation (32, 33); this
was confirmed in the present study. Our results indicate that VIP/PACAP
inhibit IL-12 (p40 and p70) production in murine peritoneal and
Raw264.7 macrophages. The inhibitory effect is
dose-dependent within a wide range of neuropeptide
concentrations (107-1010 M),
with the maximum effect being observed at 108
M.
Peritoneal macrophages have been previously shown to express VPAC1 and
PAC1 mRNA and both high and low affinity VIP/PACAP binding sites
(46, 47). Recently we showed that VPAC1 and PAC1 mRNA are expressed
constitutively, and VPAC2 expression is induced following LPS
stimulation in both peritoneal and Raw 264.7 macrophages (22, 48).
Agonist studies indicated that although both VPAC1 and VPAC2 mediate
the inhibitory effect on IL-12, the VPAC1 agonist is significantly more
efficient (75% inhibition, as compared with 25-35% for the VPAC2
agonist) (22). However, the apparent major role of VPAC1 could reflect
the balance between VPAC1 and VPAC2 expression. Because VPAC2 is
expressed relatively late during macrophage activation (12 and 24 h), VPAC1 is probably the major receptor type present during the early
culture period. The role of VPAC1 as a major player in mediating the
effect of VIP/PACAP on IL-12 is supported by the fact that a VPAC1
antagonist reverses the inhibitory effect and blocks the effect of
VIP/PACAP on both NF-B and Ets-2 complex binding and that a VPAC1
agonist mimics the effect of VIP/PACAP.
An understanding of the events mediating IL-12 secretion is complicated
by the fact that the biologically active IL-12 is a p35/p40 heterodimer
(28, 29). To secrete the biologically active heterodimer, mRNAs for
both subunits must be expressed, and both proteins must be translated
and assembled within the same cell (28). The p40 gene is transcribed
only in IL-12-producing cells, such as macrophages/monocytes, dendritic
cells, human peripheral blood mononuclear cells, and to a lesser degree
B cells. In contrast, the expression of p35 mRNA is ubiquitous and
constitutive, although free p35 polypeptide chains do not appear to be
secreted (29, 30). Cells producing the active IL-12 heterodimer also
secrete high levels of free p40 polypeptide chains, although their
biological role, particularly in humans, is still undefined (reviewed
in Refs. 23-25). These findings raise the question of how the
expression of these subunits is cooperatively modulated by either
positive or negative regulatory factors.
Previous experiments regarding VIP modulation of cytokine expression
indicated different molecular mechanisms, i.e.
transcriptional regulation for IL-2, IL-6, IL-10, and TNF
versus post-transcriptional regulation for IL-4 (10, 11, 15,
48, 49). The present study indicates that the inhibitory effect of VIP
and PACAP on IL-12 production occurs at a transcriptional level,
reducing the p40 mRNA levels, with no apparent effect on p35 gene expression.
The regulation of the IL-12 p40 gene transcription is complex and
involves multiple cis-acting elements. Transcriptional
regulation by LPS and IFN of the IL-12 p40 gene has been shown to
involve a "NF-B half-site" and transcriptional factors from the
Rel family (31). In mammalian cells the Rel family includes NF-B1
(p50), RelA (p65), c-Rel, RelB, and NF-B2 (p50B, p52) (41). NF-B consists mostly of p50/p65 heterodimers, which are complexed to the
inhibitor IB in the cytoplasm of unstimulated cells; stimuli such as
LPS and proinflammatory cytokines induce the phosphorylation and
degradation of IB, followed by the release and subsequent nuclear
translocation of the p50/p65 heterodimers, which bind to regulatory
sequences in a variety of target genes (41). The present study
indicates that VIP and PACAP inhibit NF-B binding to the IL-12 p40
promoter in IFN/LPS-stimulated Raw 264.7 cells. Similar to other
studies (31), the NF-B complex induced by LPS/IFN in macrophages
was supershifted by anti-p50, anti-p65, or anti-c-Rel Abs, suggesting
that the NF-B complexes consist of p50/c-Rel and p50/p65 and that
VIP and PACAP inhibit their nuclear translocation. We described a
similar inhibitory effect of VIP and PACAP on NF-B binding activity
for macrophage TNF and inducible nitric-oxide synthase (14, 48). In
cells treated with VIP/PACAP, the reduced NF-B binding correlates
with increased cytoplasmic IB, p65, and c-Rel levels and with
decreased nuclear p65 and c-Rel levels. It has been previously
described that the inhibition of NF-B nuclear translocation by other
anti-inflammatory agents, such as IL-11, IL-10, TGF-1,
glucocorticoids, and antioxidants, results from an increase in IB
protein levels, a decrease in IB degradation, and/or phosphorylation
(3, 50-54). In the present study, we demonstrate that VIP and PACAP
block IFN/LPS-induced reduction of IB levels by inhibiting
phosphorylation of IB subunit, without affecting mRNA
IB expression. It remains to be determined whether both
neuropeptides mediate their effects also through inhibition of IB
proteolytic degradation.
In addition to the B site, the ets element TTTCCT was identified as
a major response region in the p40 promoter in Raw 264.7 cells
(32-34). This element interacts with the large nuclear complex F1 that
binds to the region between 196 and 292 in a complex way, requiring
substantial flanking "anchoring" space (32, 33). The induction of
the F1 complex appears to correlate closely with the expression of the
IL-12 p40 gene in various cell lines and human monocytes (33, 34). F1
consists of multiple proteins including Ets-2, IRF-1, c-Rel, and a
novel 109-kDa protein (GLp109) that is induced by either LPS or IFN
(33). VIP and PACAP do not change Ets-2 binding. However, supershift
experiments indicate that both neuropeptides reduce IRF-1 and c-Rel,
without affecting the Ets-2 protein. This suggests that the inhibitory
effect of VIP/PACAP on IL-12 p40 gene expression is mediated, at least
partially, through a change in the composition of the F1-binding
complex. Indeed, the absence of either IRF-1 or c-Rel was reported to
dramatically decrease the transcriptional activation of the p40 gene
(34).
The VIP/PACAP-induced lack of c-Rel in the F1 complex could be related
to the inhibition of c-Rel nuclear translocation as discussed above. In
contrast, the reduced presence of IRF-1 in the F1 complex is probably
due to a direct inhibitory effect of VIP/PACAP on IRF-1 gene
expression. Unlike NF-B, IRF-1 is synthesized de novo
following exposure to IFN (55). In macrophages, the IRF-1 gene
responds to IFN through binding of the GAF complex generated by the
Jak1/2-STAT1 pathway (reviewed in Ref. 56). Indeed, we have recently
demonstrated that VIP and PACAP inhibit IRF-1 synthesis in macrophages
by inhibiting IFN-induced Jak1/2 activation and the subsequent STAT1
phosphorylation through a mechanism that implies an increase in the
intracellular cAMP
levels.2
In a previous study (22) we demonstrated that similar to the effect of
VIP/PACAP on TNF and inducible nitric-oxide synthase expression (14,
48), the VPAC1-mediated inhibition of IL-12 secretion in peritoneal
macrophages involves two transduction pathways, a
cAMP-dependent and a cAMP-independent pathway. In the
present study we sought to correlate the two pathways with the effects
on the IL-12 transcriptional factors. The VPAC1 and VPAC2 are coupled
primarily to the adenylate cyclase system (42), and IL-12 production is
indeed inhibited by agents that increase intracellular cAMP levels
(57-59). In the present study, forskolin and PGE2, two
cAMP-inducing agents, inhibited IL-12 production. In addition H89, a
cAMP-dependent protein kinase A inhibitor, partially
reversed the inhibitory effect of VIP/PACAP on IL-12 secretion and
reversed the VIP effect on IRF-1 associated Ets-2 binding and IRF-1
expression at both protein and mRNA levels. In contrast, H89 did
not reverse the inhibitory effect of VIP/PACAP on NF-B binding or on
the nuclear translocation of c-Rel and p65. Also, H89 did not reverse
the inhibitory effect of VIP/PACAP on reduction of cytoplasmic IB
levels. These results suggest that the cAMP-dependent
pathway mediates IRF-1 expression, whereas the cAMP-independent pathway
is responsible for the reduction in NF-B binding, presumably by
stabilizing IB. This conclusion is supported by the effects of
forskolin, a cAMP inducer. Forskolin inhibits both IRF-1 associated
Ets-2 binding and IRF-1 expression but does not affect NF-B binding
nor protein levels of c-Rel, p65, and IB. Similar observations were
made for TNF and inducible nitric-oxide synthase expression in
macrophages, where forskolin did not affect NF-B binding but changed
the composition of the CRE-binding complexes and reduced IRF-1 binding,
respectively (14, 48). The effects of cAMP on NF-B are still
debatable. For example, in some cell types, particularly thymocytes and
T cells, cAMP-elevating agents reduced NF-B binding through
stabilization of IB and subsequent impairment of p65 nuclear
translocation (60-62). In contrast, other studies reported that the
inhibition of the NF-B transcriptional activity by elevated cAMP or
by cAMP-dependent protein kinase A overexpression does not
result from an impaired nuclear translocation of the p50/p65 subunits
but from the competition between the cAMP-induced CREB and NF-B for
limited amounts of the coactivator CREB-binding protein (63, 64). VIP
and PACAP have been reported to increase CREB phosphorylation and
CREB-regulated transcription in several cell types (65-67). Therefore,
an additional mechanism in the VIP/PACAP inhibition of B-mediated
transactivation of the IL-12 gene may involve the competition between
NF-B and CREB for CREB-binding protein.
In conclusion, we have shown that the binding of VIP and PACAP to VPAC1
inhibits IL-12 production at a transcriptional level in
LPS/IFN-stimulated Raw 264.7 macrophages through two intracellular pathways. The cAMP-dependent pathway preferentially
inhibits IRF-1 mRNA expression, therefore affecting a component of
the Ets-2 transcriptional complex. The cAMP-independent pathway
inhibits the nuclear translocation of p65, a component of NF-B, and
of c-Rel, a component of both NF-B and Ets-2 transcriptional
complexes, by inhibiting the phosphorylation of IB subunit.
The biological significance of the anti-inflammatory actions of
VIP/PACAP relates to several areas of the immune response. Antigens and
microbial products in particular are potent macrophage activators that
induce the sequential release of early proinflammatory cytokines such
as TNF, IL-1, IL-6, and IL-12, followed later by anti-inflammatory
cytokines such as IL-10. TH cells, activated subsequent to macrophage
stimulation, secrete a range of pro- and anti-inflammatory cytokines
such as IL-2, IFN, IL-4, and IL-13. In normal physiological
conditions the proinflammatory cytokine cascade is down-regulated in a
timely manner by anti-inflammatory factors, such as various cytokines,
hormones, and possibly neuropeptides such as VIP and PACAP. However, in
pathological conditions such as septic shock, the activation of the
proinflammatory cytokine network becomes excessive and leads ultimately
to serious tissue damage, and possibly death. The central importance of
TNF, IL-12, and IFN in the pathogenesis of the endotoxic shock is
indicated by the fact that pretreatment with corresponding neutralizing antibodies protects against lethality (68-70). In this respect, the
ability of the neuropeptides VIP/PACAP to inhibit TNF, IL-12, and
subsequently IFN and to stimulate IL-10 production, which in turn
down-regulates TNF and IL-12, may provide a new therapeutical tool
for the down-regulation of the proinflammatory cytokine network.
Because IL-12 participates in T cell activation and CTL activity and
promotes the differentiation of TH cells into the TH1 subset (reviewed
in Refs. 23-25), VIP/PACAP might play a significant role in the
down-regulation of cell-mediated immunity in vivo. Overall
our results provide an understanding by which IL-12 is regulated by
these neuropeptides, providing the means to manipulate TH1 and TH2
responses and thereby alleviate TH1- and TH2-associated diseases.
B and Ets Activation*
§ and
TOP
ABSTRACT
INTRODUCTION
