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*
Department of Biological Sciences, Rutgers University, Newark, NJ 07102; and
Departamento Biologia Celular, Universidad Complutense, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production in LPS-stimulated macrophages. The
present study reports on the effect of VIP/PACAP on IL-10 production.
Although VIP/PACAP do not induce IL-10 by themselves, they enhance
IL-10 production in LPS-stimulated macrophages. The specific VPAC1
receptor mediates the stimulatory effect of VIP/PACAP, and cAMP is the
major second messenger involved. VIP/PACAP increase IL-10 mRNA in
LPS-stimulated cells, and the effect of transcriptional and protein
synthesis inhibitors indicates de novo IL-10 production.
Electromobility shift assays show that VIP/PACAP induce an increase in
nuclear cAMP response element (CRE)-binding complexes, with CRE binding
protein as the major active component. Treatments with either a VPAC1
antagonist or a protein kinase A inhibitor abolish IL-10 stimulation
and, concomitantly, the increase in CRE binding. Effects similar to the
in vitro stimulation of IL-10 were obtained in vivo in mice treated
with LPS and VIP or PACAP. The neuropeptides induce increased levels of
IL-10 in both serum and peritoneal fluid, and increased expression of
the IL-10 mRNA in peritoneal exudate cells. The stimulation of IL-10
production in activated macrophages represents a novel
anti-inflammatory activity of VIP and PACAP, which presumably acts
in vivo in conjunction with the inhibition of proinflammatory cytokines
such as IL-6 and TNF- to reduce the magnitude of the immune
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-1ß, IL-6, and IL-12, and immunomodulatory cytokines such as
TGF-ß1 and IL-10 (1). Since the intensity and duration of an
inflammatory process depend on the local balance between pro- and
anti-inflammatory factors, the so-called macrophage-deactivating
factors have received considerable attention lately (2, 3, 4, 5, 6).
Vasoactive intestinal peptide (VIP)3 and the pituitary
adenylate cyclase-activating polypeptide (PACAP) are two
multifunctional neuropeptides whose
primary immunomodulatory function is
anti-inflammatory in nature. VIP and PACAP have been shown to
inhibit cytokine production and proliferation in T cells (reviewed in
7 and to inhibit macrophage phagocytosis, respiratory burst, and
chemotaxis (reviewed in 8 . In agreement with their
anti-inflammatory role, VIP/PACAP were recently reported to inhibit
IL-6 and TNF- production in LPS-stimulated macrophages (9, 10, 11, 63),
and to protect mice from endotoxic shock, presumably through the
inhibition of endogenous TNF- and IL-6 (12).
The immunological actions of VIP and PACAP are exerted through a family of VIP/PACAP receptors that have been recently reclassified (13): VPAC1 and VPAC2, which exhibit similar affinities for the two neuropeptides and activate primarily the adenylate cyclase system, and PAC1, which exhibits a 300- to 1000-fold higher affinity for PACAP than for VIP and activates both the adenylate cyclase and phospholipase C systems (reviewed in 14 . Murine peritoneal macrophages were shown to possess VPAC1 and PAC1 (15, 16, 17).
IL-10, one of the major anti-inflammatory cytokines, was initially described as a Th2 product that inhibits the secretion of Th1-derived cytokines through the down-regulation of the Ag-presenting function of professional APCs (18, 19). In addition to T cells, activated monocytes/macrophages serve as a major IL-10 source, especially in response to LPS stimulation (20, 21). IL-10 inhibits several macrophage functions, such as oxidative burst, phagocytosis, nitric oxide production, and cytokine production (4, 20, 22, 23, 24, 25).
Based on their effect on macrophage function, both the neuropeptides VIP/PACAP and the cytokine IL-10 could be classified as macrophage-deactivating factors. However, in addition to their direct inhibitory effect on cytokine production by activated T cells and macrophages, VIP/PACAP may contribute to the down-regulation of the immune response through the induction of anti-inflammatory cytokines, particularly IL-10. In the present study we examine the in vitro and in vivo effects of VIP/PACAP on IL-10 protein and mRNA levels in LPS-stimulated peritoneal macrophages and investigate the molecular mechanisms involved.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthetic VIP, PACAP38, VIP1–12, and VIP10–28 were purchased from Novabiochem (Laufelfingen, Switzerland). The VPAC1 antagonist [Ac-His1,D-Phe2,K15,R16,L27]VIP3–7-GRF8–27 and the VPAC1 agonist [K15,R16,L27]VIP1–7-GRF8–27 were donated by Dr. Patrick Robberecht (Universite Libre de Bruxelles, Brussels, Belgium). The VPAC2 agonist Ro 25–1553 Ac-[Glu8,Lys12,Nle17,Ala19,Asp25,Leu26,Lys27,28,Gly29,30,Thr31]VIP- cyclo21–25 was a gift from Drs. Ann Welton and David R. Bolin (Hoffmann-La Roche, Nutley, NJ). The synthetic PAC1 agonist maxadilan was a gift from Dr. Ethan A. Lerner (Massachusetts General Hospital, Charlestown, MA). The VPAC2/PAC1 antagonist PACAP6–38, secretin, and glucagon were obtained from Peninsula Laboratories (Belmont, CA). Oligonucleotides were synthesized by the Oligonucleotide Synthesis Service of Rutgers University (Newark, NJ). Murine rIL-10 and capture and biotinylated anti-murine IL-10 mAbs were purchased from PharMingen (San Diego, CA). LPS (from Escherichia coli 055:B5), calphostin C, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS substrate), forskolin, PGE2, protease inhibitors, PMSF, EDTA, glycine, protein G-agarose, glycerol, EGTA, DTT, and avidin-peroxidase were purchased from Sigma (St. Louis, MO), and N-[2-(p-bromocinnamyl-amino)ethyl]-5-iso-quinolinesulfonamide (H89) was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Abs against CREB and c-Jun were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All reagents were dissolved in endotoxin-free distilled water and diluted in serum-free RPMI 1640 (certified as endotoxin-free; Life Technologies, Gaithersburg, MD).
Preparation of macrophages and cell cultures
Mouse peritoneal macrophages were elicited by i.p. injection of 2 ml of 4% Brewer’s thioglycolate medium (Difco, Detroit, MI) into male BALB/c mice (aged 6–10 wk; National Cancer Institute, Frederick, MD). Peritoneal exudate cells were obtained 72 h after injection by peritoneal lavage with 4 ml of 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 FCS (Atlanta Biologicals, Norcross, GA; certified as containing endotoxin levels <10 EU/ml) containing 10 mM HEPES buffer, 1 mM pyruvate, 0.1 M nonessential amino acids, 2 mM glutamine, 50 mM 2-ME, 100 U/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 x 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 morphologic and phagocytic criteria. Macrophage monolayers were incubated with RPMI complete medium and stimulated with LPS (0.5 µg/ml) in the presence or the absence of VIP or PACAP38 (from 10-12 to 10-6 M) at 37°C in a humidified incubator with 5% CO2. Cell-free supernatants were harvested at the designated time points and kept frozen (-20°C) until determination of IL-10 production by ELISA.
In vivo quantitation of IL-10 production
Male BALB/c mice (6–10 wk old) received a single i.p. dose of LPS (100 µg/mouse) in the presence or the absence of different amounts of VIP or PACAP (from 0.5–10 nmol/mouse). After different time periods (2–8 h), blood was removed through cardiac puncture, and peritoneal exudate was obtained by peritoneal lavage with 4 ml of ice-cold RPMI 1640 medium. The blood samples were allowed to clot for 1 h at room temperature; serum was obtained and kept frozen until used in IL-10 ELISA. The peritoneal suspension was centrifuged 5 min at 1800 x g, and cell-free supernatants (peritoneal fluid) were harvested and assayed for IL-10 production. The peritoneal cells were subjected to Northern blot analyses as described below.
Cytokine determination: ELISA assay for IL-10
The amounts of IL-10 present in culture supernatants, peritoneal
fluid, and serum were determined using the murine IL-10 sandwich ELISA
essentially as described previously (26), with the IL-10-specific mAb
JES3–9D7 as capture Ab and the biotinylated anti-IL-10 mAb
JES-12G8 as detection Ab. The sensitivity of the IL-10 ELISA was 10
pg/ml. The assay was specific for IL-10, with other recombinant
cytokines (IL-1, IL-2, IL-4, IL-7, IL-6, IL-12, and IFN-
) not
binding above background levels.
RNA extraction and Northern blot analysis
Northern blot analysis was performed according to standard
methods. Macrophage monolayers (107 cells/well in six-well
tissue culture plates) were cultured in 5 ml of medium and stimulated
with LPS (0.5 µg/ml) in the absence or the presence of VIP and PACAP
(10-8 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 and Schuell, Keene, NJ), and cross-linked
to the nylon membrane using UV light. The probes for murine IL-10 and
ß-actin were generated by RT-PCR as described previously (26).
Oligonucleotides were end labeled with [-32P]ATP using
T4 polynucleotide kinase. The RNA-containing membranes were
prehybridized for 16 h at 42°C and then hybridized at 60°C for
16 h with the appropriate probes. The membranes were washed twice
in 2x SSC containing 0.1% SDS at room temperature (20 min each time),
once at 37°C for 20 min, and once in 0.1x SSC containing 0.1% SDS
at 50°C (20 min). The prehybridization and hybridization buffers were
purchased from 5 Prime-3 Prime (Boulder, CO). The membranes were
exposed to x-ray films (Eastman Kodak, Rochester, NY). Signal
quantitation was performed in a PhosphorImager SI (Molecular Dynamics,
Sunnyvale, CA).
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared by a mini-extraction procedure. Briefly, peritoneal macrophages were plated at a density of 107 cells in six-well plates, stimulated, washed twice with ice-cold PBS/0.1% BSA, 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 DTT, 0.5 mM PMSF, 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 0.5% final concentration, the tubes were gently vortexed for 15 s, and nuclei were sedimented and separated from cytosol by centrifugation at 12,000 x g for 40 s. The pelleted nuclei were washed once with 0.2 ml of ice-cold buffer A, and the soluble nuclear proteins were released with 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 DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM NaN3). After incubation for 30 min on ice, the nuclear proteins were separated from the remaining nuclei by centrifugation for 10 min at 14,000 rpm at 4°C. The protein concentration in the supernatants was determined by the Bradford method, and aliquots were stored at -80°C for later use in EMSAs.
Oligonucleotides corresponding to the CRE
(5'-TTTATCCACGTCATTATGACC-3'; nucleotides
1201–1221), and NF-
B
(5'-CTCTCGGGGTTTCCTTTGGG-3'; nucleotides
1069–1088) motifs of the IL-10 promoter were synthesized (27).
Oligonucleotides were annealed by incubation for 5 min at 85°C in 10
mM Tris-HCl (pH 8.0), 5 mM NaCl, 10 mM MgCl2, and 1 mM DTT.
Fifty nanograms of the double-stranded oligonucleotides were end
labeled with [-32P]ATP using T4 polynucleotide kinase.
For EMSAs with macrophage nuclear extracts, 20,000–50,000 cpm of the
double-stranded oligonucleotide, corresponding to approximately 0.5 ng,
was used for each reaction. Binding reaction mixtures (15 µl) were
set up containing 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 DTT, 5% glycerol, and 10 mM Tris-HCl, pH 7.5). The
reaction was incubated on ice for 15 min before adding the probe,
followed by 20 min at room temperature. Samples were loaded onto 4%
nondenaturing polyacrylamide gel 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 (Clifton, NJ), drying under vacuum at
80°C, and autoradiography. In competition and Ab supershift
experiments, the nuclear extracts were incubated for 15 min at room
temperature with Ab (1 µg) or competing oligonucleotide (50-fold
excess) before addition of the labeled probe.
Statistical analysis
All values are expressed as the mean ± SD of the number of experiments. In each experiment samples were assayed in duplicate. Comparisons between groups were made using Student’s t test followed by Scheffe’s F test, with p < 0.05 as the minimum significant level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peritoneal macrophages cultured in the presence of VIP or PACAP
(from 10-7 to 10-11 M) did not secrete
detectable levels of IL-10. However, the addition of VIP/PACAP to
LPS-stimulated cultures resulted in a significant increase in IL-10
production. VIP and PACAP dose-dependently increased the amount of
IL-10 released in response to LPS, showing maximal effects at
10-6–10-8 M (Fig. 1
A). The time curves indicate
that VIP and PACAP stimulate IL-10 release as early as 4 h, with a
maximum stimulatory effect at 24 h (Fig. 1B).
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60–80% macrophages, 20–40% lymphocytes) and monolayer cell
preparations containing about 96% macrophages, confirming that the
peritoneal lymphocytes do not contribute to the IL-10 production in
response to LPS. The stimulation of IL-10 production by VIP and PACAP is mediated through VPAC1
Next we investigated whether the stimulatory effect of VIP/PACAP
could be related to the occupancy of specific receptors. First, we
compared the effect of VIP/PACAP to that of secretin, glucagon, and the
VIP and PACAP fragments VIP1–12, VIP10–28,
and PACAP6–38. IL-10 production was not affected by
different concentrations of secretin and glucagon
(10-7–10-9 M; Fig. 2A). The two VIP fragments and
PACAP6–38 failed to stimulate IL-10 production, suggesting
that intact VIP and PACAP molecules are required for their stimulatory
activity (Fig. 2A).
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B). In addition, we investigated
the ability of PACAP6–38, an antagonist specific for PAC1
and to a lesser degree for VPAC2 (32), and of a specific VPAC1
antagonist (33) to reverse the effects of VIP and PACAP. The VPAC1
antagonist reversed the effect of VIP/PACAP in a dose-dependent manner
(Fig. 2C). In contrast, PACAP6–38 did not
reverse the stimulatory effect of VIP and PACAP (Fig. 2D).
Neither the VPAC1 antagonist nor PACAP6–38 significantly
affected IL-10 levels in LPS-stimulated cells (Fig. 2, C and
D). The simultaneous addition of VIP/PACAP and the VPAC1
agonist did not result in an additive effect (Fig. 2E).
Together these results confirm the specificity of the VIP and PACAP
stimulatory effect and suggest that both neuropeptides exert their
action through VPAC1. Time course for the stimulatory effect of VIP and PACAP on IL-10 production
In the experiments described to date VIP and PACAP were added at
the same time as LPS. To determine the time interval required for the
stimulatory effect of VIP/PACAP, we stimulated peritoneal macrophages
with LPS and added 10-8 M VIP or PACAP at later times
(from 0–18 h). Supernatants were collected 24 h after the
addition of VIP/PACAP and were assayed for IL-10 production. The IL-10
levels were compared with controls containing LPS and no VIP/PACAP,
which were harvested at similar time points as the VIP/PACAP-containing
cultures. The addition of VIP and PACAP up to 12 h after LPS
stimulation resulted in significant levels of stimulation (280–355%;
Fig. 3A). Later additions (18
h) resulted in a lesser degree of stimulation (Fig. 3A). To
determine the minimum time required for the stimulatory effect of VIP
and PACAP, peritoneal macrophages were cultured with LPS in the
presence or the absence of 10-8 M VIP or PACAP. The
neuropeptides were removed by extensive washing at different times
(from 5–180 min), and the cells were cultured in medium containing LPS
without neuropeptides. Five-minute incubations resulted in significant
stimulatory effects, and 15-min incubations led to maximum stimulation
(Fig. 3B).
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To study the second messengers involved in the effect of VIP and
PACAP, we investigated the effects of calphostin C (a PKC inhibitor)
(34), H89 (a PKA inhibitor) (35), and forskolin and PGE2
(two strict cAMP-inducing agents) (36) on the release of IL-10.
Forskolin and PGE2 stimulated IL-10 production similar to
VIP and PACAP (Fig. 4A),
suggesting the involvement of cAMP. The role of cAMP as second
messenger is also supported by the effect of the PKA inhibitor H89. In
the concentration range in which calphostin C (the PKC inhibitor) does
not affect IL-10 production in LPS-stimulated cells (Fig. 4B, right panel), it does not reverse the
stimulatory effect of VIP/PACAP (Fig. 4B, left
panel). In contrast, H89 (the PKA inhibitor) reversed in a
dose-dependent manner the stimulatory effect of VIP/PACAP (Fig. 4B, center panel). These results suggest that the
stimulatory effect of VIP/PACAP on IL-10 production is mediated through
increases in intracellular cAMP.
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Having demonstrated that VIP and PACAP had a stimulatory effect on
IL-10 production, we sought to determine whether this action occurred
at a transcriptional level. We stimulated peritoneal macrophages with
LPS in the presence or the absence of 10-8 M VIP or PACAP
for 2, 4, 8, and 18 h, and total RNA was prepared and subjected to
Northern blot analysis. Although no IL-10 mRNA was detectable in
unstimulated cells (Fig. 5A),
time-dependent increasing levels of IL-10 mRNA are present in
LPS-stimulated cells (Fig. 5, B and C). At all
time points, VIP and PACAP significantly increased the levels of
specific IL-10 mRNA (Fig. 5, B and C). These
results indicate that both neuropeptides exert their actions at the
level of mRNA.
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), suggesting
that VIP and PACAP induce de novo transcription and synthesisof
IL-10.
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Although the IL-10 promoter contains a complex array of
transactivating binding sites, the cAMP-responsive element appears to
be essential for maximal IL-10 transcription (27, 37). In addition, it
has been described that cAMP-elevating drugs, such as
PGE2 and dibutyryl cAMP, up-regulate IL-10 production
by macrophages (37, 38, 39, 40). To investigate whether VIP/PACAP affect CRE
binding, we used EMSAs. Stimulation of macrophages with LPS led to a
time-dependent increase in CRE binding compared with that in
unstimulated cells, and treatment with VIP and PACAP significantly
increased this binding (Fig. 7A). The binding specificity
was confirmed using homologous (CRE) and nonhomologous (NF-B)
oligonucleotides as competitors (Fig. 7A). Ab supershift
experiments were performed to determine the composition of the
CRE-binding factors. In VIP/PACAP-treated cells, the majority of the
complex was supershifted by an anti-CREB Ab, whereas no supershift
was observed using an anti-c-Jun Ab (Fig. 7B),
indicating the presence of CREB in the CRE-binding complexes.
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C). These results suggest that the stimulation of CRE
binding by VIP is mediated through VPAC1 and the subsequent elevation
of cAMP levels. This is supported by the fact that forskolin affected
CRE binding the same way as VIP (Fig. 7C). VIP and PACAP augment IL-10 production in vivo
An attempt was made to reproduce the in vitro observations in
vivo. The i.p. injection of LPS (100 µg) resulted in elevation of
IL-10 in serum and peritoneal exudate fluid (Fig. 8A). Treatment of mice with
VIP or PACAP significantly enhanced the LPS-induced IL-10 level in
serum and peritoneal fluid (Fig. 8A). The in vivo effects of
VIP and PACAP were dose dependent, with a maximum effect at 5–10
nmol/animal (Fig. 8B). In addition, VIP and PACAP
significantly stimulated IL-10 transcription in freshly isolated
peritoneal exudate cells, 2 and 4 h after the LPS challenge (Fig. 8C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-6 (9, 11, 63). This might have clinical relevance, since
these cytokines are involved in the detrimental effects of
ischemia-reperfusion and septic shock (1, 43, 44). The present study
shows a novel property of VIP/PACAP that might contribute to their
anti-inflammatory effects, e.g., the stimulation of IL-10
production in LPS-activated macrophages. The stimulatory effect is dose
dependent within a wide range of neuropeptide concentrations
(10-6–10-10 M), with the maximum
effect being observed at 10-8 M. This is
the dose range in which VIP and PACAP modulate several other
immunological functions (7, 8, 9, 41).
The time course indicates that, similar to the effect on IL-6 and
TNF- (25, 63), VIP and PACAP affect IL-10 production rapidly, with
5- to 15-min incubations sufficient to achieve maximum stimulatory
effects. In addition, the stimulation has a refractory period of
12 h, after which the stimulatory activity of VIP/PACAP is reduced
significantly.
Of relevance is the fact that in the absence of LPS, VIP and PACAP do not stimulate IL-10 release. This observation indicates that VIP/PACAP can only amplify an endotoxin-generated signal and suggests that the in vivo action of VIP/PACAP may be restricted to cells that are actively involved in responding to Ags.
Similar to the effect on other cytokines such as IL-2, IL-6, TNF-,
and IL-12 (7, 27, 63)4 the
stimulation of IL-10 requires intact VIP/PACAP molecules. This is in
agreement with previous reports showing that either C- or N-terminal
truncations of VIP lead to significant losses in biological activity
(45, 46). Peritoneal macrophages have been previously shown to express
VPAC1 and PAC1 mRNA, and both high and low affinity VIP/PACAP binding
sites (16, 17). In addition, we have recently reported that
LPS-stimulated Raw 264.7 macrophages express mRNA for all three
VIP/PACAP receptors (47). Our agonist studies suggest that VPAC1
mediates the stimulatory effect on IL-10. This is in agreement with
Dewit et al. (11), who reported that a VPAC2 agonist has no effect on
IL-10 production in human blood monocytes. The role of VPAC1 as the
unique mediator in the effect on IL-10 production is also supported by
the fact that a VPAC1 antagonist, but not PACAP6–38, an
antagonist specific for PAC1 and to a lesser degree for VPAC2 (32),
reverses the stimulatory effect of VIP/PACAP. Also, the VPAC1
antagonist blocked the effect of VIP/PACAP on CREB binding to the CRE
site specific for the IL-10 promoter, supporting the involvement of
VPAC1 in the stimulatory effect of VIP/PACAP on IL-10 gene expression.
The VPAC1 is coupled primarily to the adenylate cyclase system (14). To fully understand the mechanism of action of VIP and PACAP, it is important to clarify which transduction pathways are involved in the stimulation of IL-10 in macrophages. It has been reported that in monocytes/macrophages IL-10 production is stimulated by cAMP-inducing agents (37, 38, 39, 40). In the present study forskolin and PGE2, two strict cAMP-inducing agents (36), stimulate IL-10 release similar to VIP and PACAP. In addition, H89, a potent and selective PKA inhibitor (35), completely reverses the effect of VIP/PACAP. In contrast, calphostin C (a specific PKC inhibitor) (34) does not alter the stimulatory action of either VIP or PACAP. These results suggest that VIP/PACAP stimulate IL-10 production in macrophages through the cAMP/PKA pathway initiated through binding to VPAC1.
Previous experiments regarding VIP modulation of cytokine expression
indicated different molecular mechanisms, i.e., transcriptional
regulation for IL-2, IL-6, and TNF- vs post-transcriptional
regulation for IL-4 (9, 47, 48, 63). The present study indicates that
the stimulatory effect of VIP and PACAP on IL-10 production occurs at
the mRNA level. This is supported by the fact that VIP/PACAP induce
increases in steady state IL-10 mRNA, and that the stimulatory effect
on the IL-10 release is inhibited by both transcriptional and protein
synthesis inhibitors. It remains to be established whether the
augmentation of steady state IL-10 mRNA levels results from an increase
in either or both the novo transcriptional rate and message
stabilization.
Although the IL-10 promoter contains a complex array of transactivating binding sites, the CRE appears essential for maximal IL-10 transcription in monocytes/macrophages (27, 37). Since VIP and forskolin induce similar increases in CRE-binding nuclear proteins, and H89 reverses the effect of VIP on CRE binding, we propose that VIP/PACAP stimulate IL-10 gene expression in peritoneal macrophages through a cAMP-dependent increase in the functionally active transcriptional factor CREB. In this respect, VIP and PACAP were reported recently to increase CREB phosphorylation and CREB-regulated transcription in several cell types (49, 50, 51). Since VIP/PACAP do not promote IL-10 production in the absence of LPS, the sole increase in CRE-binding activity is probably insufficient to induce the expression of the IL-10 gene. However, CREB could act in coordination with other transcriptional factors regulated by LPS, to induce maximal transcriptional activity for the IL-10 gene.
The necessity of several transcriptional factors acting in concert for the expression of the IL-10 gene may also explain the differences in the effects of VIP on IL-10 production in activated T cells vs activated macrophages. Previously we reported that VIP inhibits IL-10 expression in anti-CD3-stimulated T cells (26). This is in contrast to the present study, which indicates that VIP/PACAP stimulate IL-10 production in LPS-activated macrophages. In both cases, forskolin mimicked the effect of VIP, suggesting that the major transduction pathway was cAMP dependent. However, the nature of second messengers downstream from cAMP might be different in T cells and macrophages. Also, whereas in macrophages CREB induced by VIP/PACAP might act in concert with transcriptional factors induced by LPS, in T cells cAMP-dependent events such as the inhibition of JNK (52) might lead to the reduction in functionally active c-Jun protein and subsequent reduction in the transcriptional activity at the AP-1 site. Such events have been identified for the IL-2 promoter in anti-CD3-stimulated T cells treated with VIP (our unpublished observations).
The in vitro stimulatory effect of VIP/PACAP on IL-10 production
correlates with the in vivo stimulation of both IL-10 expression and
release in endotoxemic mice. VIP and PACAP both accelerate and increase
the levels of circulating IL-10 and the IL-10 mRNA expression in
peritoneal cells in endotoxemic mice. In contrast to the effect on
IL-10, VIP and PACAP inhibit both TNF- and IL-6 expression in
endotoxemic mice (9, 12, 63). Therefore, through the inhibition of
proinflammatory cytokines such as TNF- and IL-6 and the stimulation
of anti-inflammatory cytokines such as IL-10, VIP/PACAP released
within the lymphoid microenvironment may play an important role in the
down-regulation of the inflammatory response by significantly affecting
the local balance between pro- and anti-inflammatory factors. The
stimulation of the IL-10 transcription by VIP/PACAP may also have a
significant therapeutical potential, since the in vivo administration
of this anti-inflammatory cytokine leads to the prevention or
reduction of a variety of inflammatory diseases such as endotoxic shock
(53, 54, 55), viral-induced ocular inflammation (56), immune
complex-mediated lung injury (57), and chronic inflammatory bowel
disease (58).
Although VIP-ergic nerve fibers are found in most lymphoid organs, the respiratory and gastrointestinal tracts are particularly rich in VIP-containing fibers and cells (41, 59, 60), which release VIP upon electrical or chemical stimulation. Nitric oxide, for example, functions as a particularly potent signal for VIP release from enteric ganglia (61, 62). Therefore, nitric oxide produced during an inflammatory response could induce the release of high levels of VIP locally, especially in the respiratory and gastrointestinal tract, and the released neuropeptides could target the alveolar or peritoneal macrophages found in the immediate vicinity and down-regulate the production of proinflammatory agents.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Doina Ganea, Department of Biological Sciences, Rutgers University, 101 Warren Street, Newark, NJ 07102. E-mail address:
3 Abbreviations used in this paper: VIP, vasoactive intestinal peptide; PACAP, pituitary adenylate cyclase-activating polypeptide; VPAC1, type 1 VIP receptor; VPAC2, type 2 VIP receptor; GRF, growth hormone-releasing factor; H89, N-[2-(p-bromocinnamyl-amino)ethyl]-5-iso-quinolinesulfonamide; EMSA, electrophoretic mobility shift assay; CRE, adenosine 3',5'-monophosphate-responsive element; CREB, adenosine 3',5'-monophosphate-responsive element-binding protein; PAC1, pituitary adenylate cyclase-activating polypeptide receptor; PKC, protein kinase C; PKA, protein kinase A.
4 Delgado, M., E. J. Munoz-Elias, R. P. Gomariz, and D. Ganea. VIP and PACAP inhibit IL-12 production in LPS-stimulated macrophages: subsequent effect on T cell IFN- synthesis. Submitted for publication.
Received for publication August 3, 1998. Accepted for publication October 19, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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