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*
Department of Biological Sciences, Rutgers University, Newark, NJ 07102; and
Departamento Biologia Celular, Facultad de Biologia, Universidad Complutense, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, MIP-1
, monocyte chemoattractant protein 1, and RANTES in
vivo and in vitro. The inhibition of chemokine gene expression
correlates with an inhibitory effect of VIP/PACAP on NF-
B
binding and transactivating activity. The VIP/PACAP inhibition of both
chemokine production and of NF-B binding and transactivating
activity is mediated through the specific VIP receptor VPAC1, and
involves both cAMP-dependent and -independent intracellular pathways.
In an in vivo model of acute peritonitis, the inhibition of chemokine
production by VIP/PACAP leads to a significant reduction in the
recruitment of polymorphonuclear cells, macrophages, and lymphocytes
into the peritoneal cavity. These findings support the proposed role of
VIP and PACAP as key endogenous anti-inflammatory agents and
describe a novel mechanism, i.e., the inhibition of the production of
macrophage-derived chemokines. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, IL-1, IL-12, and NO, which contribute to
pathophysiological changes associated with several acute and chronic
inflammatory conditions (1), and inflammatory chemokines
that recruit and activate blood-derived leukocytes (2, 3, 4, 5, 6).
The chemokine superfamily consists of low-m.w. polypeptides that are
categorized into four subfamilies, CXC, CC, C, and
CX3C, based on the arrangement of positionally
conserved cysteine motifs within the N terminus. The CXC and CC
chemokines predominate and, thus, have been the most extensively
studied. The great interest generated by the discovery of chemokines
lies in their specificity; for example, the CXC chemokines IL-8 and
macrophage inflammatory protein
(MIP)3
-2 activate and induce the directional migration of neutrophils, whereas
CC chemokines, including MIP-1, MIP-1, monocyte chemoattractant
protein (MCP)-1, and RANTES, are chemotactic for monocytes/macrophages
and T cells (2, 3, 4, 5, 6).
Vasoactive intestinal peptide (VIP) and the structurally related
peptide, the pituitary adenylate cyclase-activating polypeptide
(PACAP), are two neuropeptides present in the immune microenvironment
(7) that elicit a broad spectrum of biological functions,
including actions on innate and adaptive immunity (7, 8, 9).
Although VIP and PACAP affect a variety of immune functions, their
primary immunomodulatory function is anti-inflammatory in nature.
VIP and PACAP have been shown to inhibit cytokine production and
proliferation in T cells (10), and to inhibit several
macrophage functions, including phagocytosis, respiratory burst, and
chemotaxis (11), as well as LPS-induced IL-6, TNF-,
IL-12, and NO production (8, 9). In agreement with their
anti-inflammatory role, VIP/PACAP recently were reported to protect
mice from lethal endotoxemia, presumably by down-regulating endogenous
proinflammatory macrophage-derived mediators (12).
Because chemokines are involved in controlling the nature and magnitude of the inflammatory response, in this study, we examined the in vitro and in vivo effects of VIP and PACAP on CXC and CC chemokine production in activated peritoneal macrophages, and how the VIP/PACAP regulation of chemokine expression is functionally linked to leukocyte migration. This study further clarifies the role played by VIP and PACAP in the attenuation of the inflammatory response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthetic VIP and PACAP38 were purchased from Calbiochem-Novabiochem (Laufelfingen, Switzerland). The PAPAC receptor (PAC)1/VIP receptor (VPAC) 2 antagonist PACAP6–38 was obtained from Peninsula Laboratories (Belmont, CA). 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 Patrick Robberecht (Universite Libre de Bruxelles, Brussels, Belgium). The VPAC2 agonist Ro25-1553 Ac-[Glu8, Lys12, Nle17,Ala19, Asp25, Leu26, Lys27,28, Gly29,30, Thr31]-VIP cyclo21–25 was a generous gift from A. Welton and D. R. Bolin (Hoffmann-La Roche, Nutley, NJ). The synthetic PAC1 agonist maxadilan was a generous gift from E. A. Lerner (Massachusetts General Hospital, Charlestown, MA). Capture and biotinylated Abs against murine KC (IL-8), RANTES, MIP-2, and MCP-1 were purchased from PeproTech (Rocky Hill, NJ). LPS (from Escherichia coli 055:B5), DEAE-dextran, protease inhibitors, dibutyryl-cAMP (db-cAMP), and forskolin 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 p65, p50, and CREB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Murine rIL-10 and neutralizing anti-IL-10 Abs were purchased from BD PharMingen (San Diego, CA).
Preparation of peritoneal macrophages and cell cultures
Mouse peritoneal macrophages were elicited by i.p. injection of 2 ml of 4% Brewer’s thioglycollate medium (Difco, Detroit, MI) into male BALB/c mice (aged 6–10 wk). 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 FCS (Life Technologies, Rockville, MD), and were seeded in flat-bottom 96-well microtiter plates (Corning Glass, Corning, NY) at 8 x 104 cells per well in a final volume of 200 µl. The cells were incubated at 37°C for 2 h to adhere, and nonadherent cells were removed by repeated washing with RPMI 1640 medium. At least 96% of the adherent cells were Mac-1+ macrophages.
Raw 264.7 mouse macrophage cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with 10% FCS. The cells were plated in flat-bottom 96-well microtiter plates at 8 x 104 cells per well in a final volume of 200 µl for 24 h, followed by two washings with DMEM medium.
Macrophage monolayers were stimulated with 10 ng/ml LPS in the presence or 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 chemokine determination by ELISA.
Plasmids, transfections, and luciferase assay
NF-B-dependent gene expression was evaluated with a
luciferase reporter gene driven by four tandem copies of the k enhancer
(B4) in a pUC vector (Clontech, Palo Alto,
CA). The plasmid pRc/RSV-p65 containing the entire cDNA of p65 was
provided G. J. Nabel and J. Stein through the National Institutes of
Health AIDS Research and Reference Reagent Program. Empty vectors
pRc/RSV and pUC-18 (Invitrogen, Carlsbad, CA) were used to keep
constant total transfected DNA concentration in each experiment. To
assess variations in transfection efficiencies, cells were transfected
with 2 µg of the control plasmid pCH110 (Amersham Pharmacia Biotech,
Piscataway, NJ), which expresses the LacZ gene. Levels of
-galactosidase were determined by the Galacto-Light assay system
(Tropix, Bedford, MA) and exhibited <15% variation between
samples.
Raw 264.7 cells were transiently transfected with a total of 10–30
µg of plasmid DNA by the DEAE-dextran procedure as described
previously (13). Twenty-four hours after transfection,
cells were stimulated with LPS (10 ng/ml) in the absence or presence of
difference concentrations of VIP or PACAP as indicated above. After
6 h of incubation, luciferase assays were conducted according to
the instructions of the manufacturer (Promega, Madison, WI). Light
emission was measured in a luminescence microplate counter (Top-Count;
Packard, Meriden, CT). Luciferase activity, expressed in arbitrary
light units, was corrected for protein concentration or normalized to
coexpressed -galactosidase levels.
EMSA
Nuclear extracts were prepared as described previously
(14). Double-stranded oligonucleotides (50 ng)
corresponding to the NF-B sites from murine MCP-1
(5'-ACTGCCCTCAGAATGGGAATTTCCACGCTCTTATC-3'; Ref.
15), RANTES
(5'-TTTTGGAAACTCCCCTTAGGGGATGCCCCTCA-3';
Ref. 16), MIP-2
(5'-CCCTGAGCTCAGGGAATTTCCCTGGTCCCCG-3'; Ref.
17), and KC (5'-TACTCCGGGAATTTCCCTGGCC-3';
Ref. 18), were end-labeled with [
-32P]
ATP by using T4 polynucleotide kinase. For EMSAs with Raw 264.7
nuclear extracts, 20,000–50,000 cpm of double-stranded
oligonucleotides, corresponding to 
0.5 ng, were used for each
reaction. The 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 mixtures were
incubated on ice for 15 min before adding the probe, followed by for
another 20 min 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 Ab supershift experiments, the
nuclear extracts were incubated for 15 min at room temperature with the
specific Ab (1 µg) or competing cold oligonucleotide (50-fold excess)
before the addition of the labeled probe.
Model of acute inflammation: LPS peritonitis
Male BALB/c mice (6–10 wk old) received a single i.p. dose of
LPS (25 µg/mouse, 1.2–1.4 mg/kg) in the presence or absence of
different amounts of VIP or PACAP (from 0.5 to 10 nmol/mouse). After
different time periods (1 to 48 h), blood was removed through
cardiac puncture, and peritoneal exudate was obtained by peritoneal
lavage with 3 ml of ice-cold PBS containing 3 mM EDTA. The blood
samples were allowed to clot for 1 h at room temperature, and
serum was obtained and kept frozen until used in chemokine ELISA. The
peritoneal suspension was centrifuged 5 min at 1800 x
g, and cell-free supernatants (peritoneal fluid) were
harvested and assayed for chemokine production. Peritoneal cells were
counted and adjusted in PBS/3 mM EDTA medium at 3 x
106 cells/ml. The number of viable cells in the
different peritoneal subpopulations was determined by flow cytometry
(FACScan; BD Biosciences, Mountain View, CA). Briefly, peritoneal
lymphocytes, macrophages, and polymorphonuclear cells (PMN) were gated
according to their different forward scatter and side scatter
characteristics and counted. Alternatively, aliquots of the lavage
fluid were stained with Turk’s solution (0.01% crystal violet in 3%
acetic acid), and differential cell counts were performed by light
microscopy. The large predominance of neutrophils (99%) in the PMN
population was confirmed in cytospin preparations stained with
May-Grunwald and Giemsa.
Chemokine ELISA
The content of chemokines in the culture supernatants, serum,
and peritoneal fluid was determined by specific sandwich ELISAs as
recommended by the manufacturer. The lower limit of detection for
MCP-1, MIP-1, MIP-1, MIP-2, RANTES, and KC was 16, 5, 7,
8, 10, and 15 pg/ml, respectively.
RNase protection assay (RPA) for the detection of chemokine mRNA expression
Peritoneal macrophages were cultured at a concentration of
2 x 106 cells/ml in 100-mm tissue culture
dishes and stimulated with LPS (10 ng/ml) in the presence or absence of
VIP (10-8 M) or PACAP
(10-8 M) for up to 12 h. Cells were
collected at different time points (0 and 12 h) and total RNA was
isolated with the Ultraspec RNA reagent (Biotecx Laboratories, Houston,
TX) as recommended by the manufacturer. RPA were performed on 2.5–5
µg of RNA with the Riboquant MultiProbe RPA system (BD PharMingen)
following the manufacturer’s instructions. Each commercial kit
contained a set of chemokine templates as well as a template for the
housekeeping gene, GAPDH. [-32P]UTP-labeled
antisense RNA probes were synthesized by in vitro transcription from
these cDNA templates. Antisense RNA probes were purified by
phenol/chloroform extraction and ethanol precipitation and were
hybridized with the RNA samples at 56°C overnight. Unhybridized
single-stranded RNA was digested by RNase treatment. Double-stranded
RNA was purified by phenol/chloroform extraction and ethanol
precipitation. The samples were electrophoresed on a 5% denaturing
polyacrylamide gel. The gel then was dried and exposed to x-ray films
(Kodak, Rochester, NY). Signal quantitation was performed in a
PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the effect of VIP and PACAP on endotoxin-induced
chemokine production, peritoneal macrophages were stimulated with
different concentrations of LPS in the absence or presence of various
doses of VIP or PACAP. The amounts of various released chemokines were
assayed by ELISA. Unstimulated macrophages produce very low amounts of
chemokines (Fig. 1
A). Treatment with LPS results in a time-dependent increase
in the production of the CXC chemokines MIP-2 and KC, as well as of the
CC chemokines MIP-1, MIP-1, MCP-1, and RANTES, with peak levels
between 12 and 24 h (Fig. 1A). In general, CXC and CC
chemokine levels remain high, with only a gradual decline between 24
and 48 h. VIP and PACAP inhibit in a dose- and time-dependent
manner the production of the CXC and CC chemokines tested (Fig. 1).
This inhibition occurred as early as 2 h, with a maximum effect at
24 h (Fig. 1A). Moreover, the reduction of chemokine
production was maintained throughout 72 h (data not shown),
indicating that VIP/PACAP do not delay, but rather reduce chemokine
release. The dose-response curves were similar for VIP and PACAP,
showing maximal effects around 10-8 M (Fig. 1B). The inhibitory effect of both neuropeptides was
observed over a wide range of LPS concentrations, showing maximal
effect at 1–10 ng/ml LPS (Fig. 1C).
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Because the highest inhibition was observed for macrophages stimulated with 10 ng/ml LPS, at a neuropeptide concentration of 10-8 M after 24 h of culture, we used these conditions in the rest of the experiments.
Inhibition of chemokine production by VIP and PACAP is mediated through VPAC1
Next we investigated whether the inhibitory effect of VIP/PACAP
could be related to occupancy of specific receptors. The immunological
actions of VIP and PACAP are exerted through a family of receptors
consisting of VPAC1, VPAC2, and PAC1 (19). Peritoneal
macrophages express VPAC1 and PAC1 mRNA constitutively, and VPAC2 mRNA
is inducible on LPS stimulation (20). To determine which
of the VIP/PACAP receptors are involved in the inhibition of chemokine
production, we used specific receptor agonists and antagonists. We
investigated the effect of a VPAC1 agonist (21), a VPAC2
agonist (Ro 25-1553; Ref. 22), and of maxadilan, a
specific PAC1 agonist (23) on LPS-induced chemokine
production. The VPAC1 agonist, but not the VPAC2 and PAC1 agonists,
inhibited the release of all CXC and CC chemokines assayed with a
potency similar to that of VIP/PACAP (Fig. 2A). In addition, we investigated the ability of
PACAP6–38, an antagonist specific for PAC1 and,
to a lesser degree for VPAC2 (24), and of a specific VPAC1
antagonist (25), to reverse the effects of VIP and PACAP.
Increasing concentrations of the antagonists
(10-8 to 10-5 M) were
added simultaneously with a fixed concentration of VIP or PACAP
(10-8 M). The VPAC1 antagonist reversed the
effect of VIP/PACAP in a dose-dependent manner (Fig. 2B). In
contrast, PACAP6–38 did not reverse the
inhibitory effect of VIP and PACAP (Fig. 2B). Together,
these results suggest that VIP and PACAP exert their action primarily
through VPAC1.
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To determine whether intracellular cAMP mediates the inhibitory
effect of VIP/PACAP on chemokine production, we determined the effects
of calphostin C (a protein kinase C inhibitor), H89 (a protein kinase A
(PKA) inhibitor), forskolin (a strict cAMP-inducing agent), and db-cAMP
(a cAMP analog). Forskolin and db-cAMP inhibit production of all
chemokines in LPS-stimulated macrophages, although they show less of an
effect at lower concentrations (0.1 and 10 nM), compared with VIP and
PACAP (Fig. 3A). The involvement of cAMP also is supported by the effects
of the two protein kinase inhibitors. In contrast to calphostin C, H89
partially reverses the inhibitory effect of VIP/PACAP (Fig. 3B). These results suggest that the inhibitory effect of
VIP/PACAP is partially mediated through increases in intracellular
cAMP.
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Because VIP/PACAP enhance IL-10 production in LPS-stimulated
macrophages (26) and IL-10 was shown to inhibit chemokine
production (27), we investigated whether the inhibitory
effect of VIP/PACAP is mediated through IL-10. LPS-stimulated
macrophages treated with murine rIL-10 expressed reduced levels of
MIP-1, RANTES, MCP-1, and MIP-2, and anti-IL-10 Abs (but not
control Abs) enhanced chemokine production (Fig. 4). However, the anti-IL-10 Abs did not reverse the inhibitory
effect of VIP or PACAP on chemokine production (Fig. 4), indicating
that the effect of VIP/PACAP is not mediated through IL-10.
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Peritoneal macrophages were stimulated with LPS in the presence or
absence of 10-8 M VIP or PACAP for 2, 6, 12, 18,
and 24 h, and total RNA was subjected to RPA analysis.
Progressively increased levels of MIP-2, MIP-1, MIP-1, MCP-1, and
RANTES mRNA are present in LPS-stimulated cells (up to 6–12 h; Fig. 5). At all time points, VIP and PACAP significantly inhibited the levels
of both CXC and CC chemokine mRNA, with a maximum effect at 6–12 h
(Fig. 5). The amount of mRNA correlated with the release of chemokine
proteins in replica dishes (data not shown).
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B binding and subsequent
NF-B-dependent gene activation
Although the promoters of most of CXC and CC chemokines contain
complex arrays of transactivating binding sites, NF-B
ap-pears to be essential for maximal chemokine transcription after LPS
stimulation (15, 16, 17, 18, 28, 29, 30). We have reported previously
that VIP and PACAP inhibit production of several proinflammatory
cytokines by activated macrophages by down-regulating NF-B binding
and activation (13, 31, 32, 33). To investigate whether
VIP/PACAP affect NF-B binding, we used nuclear extracts from
LPS-stimulated peritoneal macrophages treated with or without VIP or
PACAP. VIP and PACAP inhibited the binding to B sites from the KC,
MIP-2, MCP-1, and RANTES promoters (Fig. 6). The binding specificity was tested with specific (B) and
nonspecific (cAMP response element) competitors, and the composition of
the binding complexes (p65/p50) was determined by supershift assays
(Fig. 6). Because peritoneal macrophages exhibited low transfection
efficiencies, we used the murine macrophage cell line Raw 264.7. First,
we confirmed that VIP and PACAP affect chemokine production in the Raw
cells similar to peritoneal macrophages. Indeed, VIP and PACAP inhibit
in a dose- and time-dependent manner the production of KC, MIP-2,
MIP-1, MIP-1, MCP-1, and RANTES (Fig. 7A). Similar to peritoneal macrophages, stimulation of Raw
cells with LPS led to an increase in NF-B binding to the B sites
from the KC, MIP-2, MCP-1, and RANTES promoters, and treatment with VIP
and PACAP significantly inhibited this binding (Fig. 7B).
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B transactivating
activity, Raw 264.7 cells were transiently transfected with a
(B)4-luciferase reporter plasmid, and 48
h later, the cells were stimulated with LPS in the presence or absence
of VIP or PACAP and assayed for NF-B-dependent transcription 5
h later. LPS stimulation leads to an 18-fold increase in NF-B
transcriptional activity (Fig. 7
C). Treatment with VIP or
PACAP strongly inhibits LPS-induced NF-B activity (Fig. 7C). The inhibitory effect is dose-dependent, with a maximum
at 10 nM VIP or PACAP (Fig. 7C). These effects are similar
to those observed with the human monocytic cell line THP-1
(13).
Receptors and intracellular pathways involved in the inhibitory
activity of VIP and PACAP on the NF-B complex
Because the inhibitory effect of VIP on chemokine production is
mediated primarily through VPAC1 and cAMP, we determined the effect of
the VPAC1 antagonist and of the PKA inhibitor H89 on the changes
induced by VIP in B-binding complexes and NF-B-dependent gene
activation. The inhibitory activity of VIP on LPS-mediated NF-B
binding was completely reversed by the VPAC1 antagonist (Fig. 8A, lane 3), but not by H89 (Fig. 8A, lane
4). These results suggest that the inhibition of NF-B binding
by VIP is mediated through VPAC1, but is mostly cAMP-independent. This
is supported by the fact that forskolin (a cAMP inducer) does not
significantly affect NF-B binding (Fig. 8A, lane 5).
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B transcriptional activity is completely
reversed by the VPAC1 antagonist, and partially reversed by increasing
concentrations of H89 (Fig. 8B). In addition, forskolin
mimics partially the effect of VIP (Fig. 8B). These findings
suggest that the effect of VIP on NF-B transactivating activity is
mediated through VPAC1 and involves both cAMP-dependent and
cAMP-independent pathways. In vivo inhibition of endotoxin-induced chemokine production by VIP and PACAP
An attempt was made to reproduce the previous observations in
vivo. Injection i.p. of LPS (25 µg) resulted in the transient
elevation of KC, MIP-2, MIP-1, MIP-1, MCP-1, and RANTES in serum
and peritoneal exudate fluid (Fig. 9). Simultaneous treatment of mice with VIP or PACAP significantly
reduced the in vivo LPS-induced CXC and CC chemokines (Fig. 9). The
effects of VIP and PACAP were dose-dependent, with a maximum effect at
5–10 nmol/mouse (data not shown).
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Chemokines are chemoattractants for blood-derived
leukocytes, and there is a clear link between chemokine production and
leukocyte recruitment in acute inflammatory models (2, 3, 4, 5, 6).
We investigated whether VIP and PACAP also are capable of inhibiting
leukocyte recruitment in a murine model of acute inflammation.
Injection i.p. of LPS induces a time-dependent accumulation in the
peritoneal cavity of various cell populations. PMN, predominantly
neutrophils, accumulate first, with a maximal influx at 12 h after
endotoxin administration (Fig. 10). Macrophages and lymphocytes are slightly delayed, starting to
accumulate between 12 and 18 h, peaking at 24 h, and being
still significantly elevated at 48 h (Fig. 10). Treatment with VIP
or PACAP (5 nmol/mouse) significantly reduced LPS-induced PMN,
macrophage, and lymphocyte influx into the peritoneal cavity (Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-12, NO, and IFN-
(12).
The present study demonstrates a novel property of VIP/PACAP that might
contribute to their anti-inflammatory effects. VIP and PACAP
inhibit the production of the proinflammatory CXC chemokines KC and
MIP-2, and of the CC chemokines MIP-1, MIP-1, MCP-1, and RANTES
by LPS-stimulated macrophages. The inhibitory effect is dose-dependent
within a wide range of neu-ropeptide concentrations
(10-6–10-10 M), with a
maximum effect at 10-8 M. This is the dose range
in which VIP and PACAP modulate several other immunological functions
(8, 9, 10, 11).
Because the anti-inflammatory action of VIP and PACAP is mediated primarily through a direct effect on macrophages (8, 9), and because LPS-stimulated macrophages elicit the proinflammatory cascade associated with sepsis (34, 35), we focused on chemokine production by LPS-stimulated peritoneal macrophages.
Both peritoneal macrophages and the Raw 264.7 cell line express VPAC1
and PAC1 mRNA constitutively and VPAC2 mRNA after LPS-stimulation
(20, 31). Our agonist studies suggest that VPAC1 mediates
the inhibitory effect on CXC and CC chemokine production. The role of
VPAC1 as the unique mediator in the effect on CK production also is
supported by the fact that a VPAC1 antagonist, but not
PACAP6–38, an antagonist specific for PAC1 and
to a lesser degree for VPAC2, reverses the inhibitory effect of
VIP/PACAP. Also, the VPAC1 antagonist blocked the effect of VIP/PACAP
on NF-B binding to the B site specific for the KC, RANTES,
MIP-1 and MCP-1 promoters, supporting the involvement of VPAC1 in
the inhibitory effect of VIP/PACAP on chemokine gene expression.
The VPAC1 is coupled primarily to the adenylate cyclase system
(19), and production of some chemokines (i.e., MCP-3) is
indeed inhibited by agents that increase intracellular cAMP levels
(38). In the present study, forskolin and
PGE2, two cAMP inducing agents, inhibited
chemokine production. In addition, H89, a potent and selective PKA
inhibitor reversed the inhibitory effect of VIP/PACAP, supporting the
involvement of the cAMP/PKA pathway. However, because reversal was
incomplete, a second cAMP-independent pathway may participate in the
transduction of the VIP/PACAP signal. Similar observations were made
previously for the inhibitory effect of VIP/PACAP on TNF-, IL-12, NO
production in macrophages (20, 31, 32, 33), and on IL-2 and
IL-10 production in lymphocytes (39). The existence of a
second, cAMP-independent pathway, is supported also by the fact that at
concentrations that are physiologically relevant for VIP
(10-8 M) the peptide induces less cAMP than
forskolin (32) while acting as a more potent chemokine
inhibitor. The nature of this second transduction pathway remains to be
determined.
Chemokine synthesis is controlled at several levels. Whereas
posttranscriptional, translational, and posttranslational mechanisms
play important roles, chemokine transcription appears to be the primary
regulatory site. The present study indicates that the inhibitory effect
of VIP and PACAP on CXC and CC chemokine production occurs through
reduction in mRNA levels. How can VIP and PACAP regulate such a wide
spectrum of chemokine genes? The answer is probably that gene
expression for many chemokines depends on the pleiotropic transcription
factor NF-B (15, 16, 17, 18, 28, 29, 30). 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 (40). The present study indicates
that VIP and PACAP inhibit LPS-induced NF-B/DNA binding to the B
motifs in the promoters of KC, RANTES, MCP-1, and MIP-1. In
addition, VIP/PACAP reduce NF-B transactivating activity. The effect
on NF-B/DNA binding is cAMP-independent, whereas the inhibition of
the NF-B transactivating activity is mediated through both a
cAMP-dependent and -independent pathway. This is in agreement with
previous observations (13, 31, 32, 33), and is presumably
attributable to the fact that NF-B transactivation involves several
steps in addition to NF-B DNA binding. In LPS-stimulated human
monocytes, we showed that VIP/PACAP act at multiple levels
(13). First, VIP/PACAP inhibit p65 nuclear translocation
and NF-B DNA binding by stabilizing the inhibitor IB in a
cAMP-independent manner. Second, VIP/PACAP induce the phosphorylation
of CREB, which binds the coactivator CREB-binding protein (CBP), found
in limiting amounts in the nucleus. This results in a decrease in
p65/CBP complexes, which further reduces NF-B transactivation.
Third, VIP and PACAP inhibit the mitogen-activated
protein/extracellular signal-related kinase kinase
kinase1/mitogen-activated protein/extracellular signal-related kinase
kinase 3/6/p38 pathway, ultimately affecting the phosphorylation of the
coactivator, TATA-box binding protein (TBP), resulting in a reduction
in TBP binding to both p65 and the TATA box. In contrast to the effect
on p65 nuclear translocation, the effects on CBP and TBP are mediated
through the cAMP/PKA pathway (13).
IL-10 was reported to inhibit cytokine and chemokine production in
alveolar macrophages and microglia by preventing NF-B nuclear
translocation (27, 41, 42). We showed previously that VIP
and PACAP enhance IL-10 production in LPS-stimulated peritoneal
macrophages (26). Therefore, the effects of VIP/PACAP on
chemokine expression might be mediated through IL-10. However, this
doesn’t seem to be the case, because the addition of neutralizing
anti-IL-10 Abs did not reverse the inhibitory effect of
VIP/PACAP.
Because the release of chemokines results in the recruitment of
blood-derived leukocytes (2, 3, 4, 5, 6), the fact that VIP and
PACAP inhibit chemokine production in vivo is of obvious biological
significance. In agreement with previous reports (43, 44),
we found that i.p. administration of LPS results in a rapid production
of chemokines. KC and MIP-2 both exhibited early (2 h) peak levels in
peritoneum, consistent with their role in the recruitment of
neutrophils, the first leukocyte population to arrive at a site of
inflammation. Chemokines responsible for recruiting subsequent
leukocyte infiltrates, i.e., monocytes/macrophages and T lymphocytes
(i.e., MCP-1, MIP-1, RANTES, and MIP-1), peaked at a later time
point (4 h). VIP and PACAP prevented the influx and accumulation into
the peritoneal cavity of neutrophils, macrophages, and lymphocytes,
presumably through the inhibition of chemokine production. These
findings support the proposed role for VIP and PACAP as key
anti-inflammatory agents in vivo, and suggest an additional
molecular mechanism, i.e., the inhibition of macrophage-derived
chemokine production.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Doina Ganea, Rutgers University, Department of Biological Sciences, 101 Warren Street, Newark, NJ 07102. E-mail address: dganea{at}andromeda.rutgers.edu
3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; VIP, vasoactive intestinal peptide; PACAP, pituitary adenylate cyclase-activating polypeptide; PAC, PACAP receptor; VPAC, VIP receptor; db-cAMP, dibutyryl cAMP; PMN, polymorphonuclear cell(s); RPA, RNase protection assay; PKA, protein kinase A; CBP, CREB-binding protein; TBP, TATA-box binding protein.
Received for publication January 18, 2001. Accepted for publication May 2, 2001.
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), and lymphocytes
(Lymph) were determined by flow cytometry as described in
Materials and Methods. Data are the mean ± SD of
three experiments.