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B and IFN Regulatory Factor 1 Activation1
*
Department of Biological Sciences, Rutgers University, Newark, NJ 07102; and
Departamento Biologia Celular, Universidad Complutense, 28040 Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-, and
LPS/IFN--stimulated peritoneal macrophages and the Raw 264.7 cell
line. Both VIP and PACAP inhibit NO production in a dose- and
time-dependent manner by reducing iNOS expression at protein and mRNA
level. VPAC1, the type 1 VIP receptor, which is constitutively
expressed in macrophages, and to a lesser degree VPAC2, the type 2 VIP
receptor, which is induced upon macrophage activation, mediate the
effect of VIP/PACAP. VIP/PACAP inhibit iNOS expression and activity
both in vivo and in vitro. Two transduction pathways appear to be
involved, a cAMP-dependent pathway that preferentially inhibits IFN
regulatory factor-1 transactivation and a cAMP-independent pathway that
blocks NF-
B binding to the iNOS promoter. The down-regulation of
iNOS expression, together with previously reported inhibitory effects
on the production of the proinflammatory cytokines IL-6, TNF-
, and
IL-12, and the stimulation of the anti-inflammatory IL-10, define
VIP and PACAP as "macrophage deactivating factors" with significant
physiological relevance. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and LPS represent one of the most
potent combinations of synergizing stimuli (3). Once synthesized, iNOS
is responsible for prolonged, high-output production of NO. Induced NO
production is one of the principal mechanisms of macrophage
cytotoxicity for tumor cells, bacteria, protozoa, helminthes, and fungi
(1, 4). In general, expression of iNOS follows a generalized or
localized inflammatory response resulting from infection or tissue
injury. Despite its beneficial role in host defense, sustained NO
production can be deleterious to the host, and NO synthesis induced by
cytokines and/or inflammatory stimuli has been implicated in
experimental arthritis, inflammatory bowel disease, hypotension
associated with septic shock, and other types of tissue injury (5, 6, 7, 8).
Therefore, the selective inhibition of expression of this enzyme
represents an important therapeutic goal.
Vasoactive intestinal peptide (VIP) and 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
attenuate reperfusion injury following ischemia of brain and lung
(8, 9, 10), to inhibit T cell proliferation and cytokine production
(reviewed in 11), and to inhibit several macrophage functions,
such as phagocytosis, respiratory burst, chemotaxis (reviewed in 12), and LPS-induced IL-6 and TNF- production (13, 14, 15, 16). Furthermore,
we have recently demonstrated that VIP and PACAP protect mice from
lethal endotoxemia, presumably by reducing the levels of endogenous
TNF- and IL-6 (17).
The functional relationship between VIP and NO is rather complicated. VIP and NO can be colocalized and coreleased from some neurons and can regulate each other’s release in some tissues (reviewed in 8). In some physiological outcomes, such as the relaxation of smooth muscle, VIP and NO cooperate, whereas in others, such as the inflammatory response, they play opposite roles, with NO increasing and VIP defending against tissue and cell injury. The opposite role played by VIP and NO in an inflammatory process raises the possibility that VIP may regulate NO production or activity. Indeed, in injury models that involve neuronal NOS, VIP does not inhibit NO synthesis, but prevents its toxic action (18, 19). However, much less is known about the role of VIP in the expression or activity of iNOS, the macrophage-specific NOS, in response to inflammation.
To further clarify the role played by VIP and PACAP in the attenuation of the inflammatory response, in this study we examine the in vitro and in vivo effects of both neuropeptides on NO production and iNOS transcription in activated peritoneal macrophages. We investigate the molecular mechanisms involved, including the specific receptors, the intracellular signal pathways, and the nuclear transactivating factors that mediate the effect of VIP/PACAP on NO synthesis.
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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 type 1 VIP receptor (VPAC1) antagonist
[Ac-His1, D-Phe2, K15,
R16, L27] VIP (3, 4, 5, 6, 7)-GRF (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) and the VPAC1
agonist [K15, R16, L27] VIP
(1, 2, 3, 4, 5, 6, 7)-GRF (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) were kindly donated by Dr. Patrick Robberecht
(Universite Libre de Bruxelles, Belgium). The VPAC2 agonist Ro 25-1553
Ac-[Glu8, Lys12,
Nle17,Ala19, Asp25,
Leu26, Lys27,28, Gly29,30,
Thr31]-VIP cyclo (21, 22, 23, 24, 25) was a generous gift from Drs.
Ann Welton and David R. Bolin (Hoffmann-La Roche, Nutley, NJ). The
synthetic PACAP receptor (PAC1) agonist maxadilan was a generous gift
from Dr. Ethan A. Lerner (Massachusetts General Hospital, Charlestown,
MA). 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
IFN- was purchased from PharMingen (San Diego, CA). LPS (from
Escherichia coli 055:B5), calphostin C, forskolin,
prostaglandin E2 (PGE2),
L-arginine, NADPH, flavin-adenine dinucleotide,
tetrahydrobiopterin, L-lactic dehydrogenase, sodium
pyruvate, sulfanilamide, N-[naphthyl]ethyl-enediamine
dihydrochloride, protease inhibitors, PMSF, EDTA, glycine, protein
G-agarose, glycerol, EGTA, and DTT were purchased from Sigma (St.
Louis, MO), and
N-[2-(p-bromocinnamyl-amino)
ethyl]-5-iso-quinolinesulfonamide (H89) from ICN Pharmaceuticals
(Costa Mesa, CA). Abs against IFN regulatory protein-1 (IRF-1) and
NF-B (p50 and p65) were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA).
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, Grand Island, NY), 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 per 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.
Raw 264.7 mouse macrophage cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 10 µg/ml streptomycin, and 10% FCS (complete culture medium). The cells were plated in flat-bottom 96-well microtiter plates for 24 h. Nonadherent cells were removed by aspiration and two washings with DMEM medium.
Macrophage monolayers (murine peritoneal macrophages and Raw 264.7
cells) were incubated with complete medium and stimulated with
0.5 µg/ml LPS or/and 200 U/ml IFN- in the presence or
absence of VIP or PACAP38 (from 10-12 to 10-7
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 NO determination.
Determination of NO
The amount of NO formed was estimated from the accumulation of the stable NO metabolite nitrite by the Griess assay (20). Equal volumes of culture supernatants (90 µl) and Griess reagents (90 µl of 1% sulfanilamide/0.1% N-[naphthyl]ethyl-enediamine dihydrochloride in 2.5% H3PO4) were mixed, and the absorbance was measured at 550 nm. The amount of nitrite was calculated from a NaNO2 standard curve.
Determination of NO synthase activity
Lysates from activated macrophages were prepared, and the NO
synthase activity was measured as described (21). Murine peritoneal
macrophages and Raw 264.7 cells were activated with IFN-/LPS in the
presence or absence of VIP or PACAP and harvested at 4°C. Cells were
lysed by three cycles of freeze/thaw in 40 mM Tris-HCl, pH 8.0, 0.1 mM
EGTA, 0.1 mM EDTA, 12 mM 2-ME, and protease inhibitors (0.1 mM PMSF, 5
µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml chymostatin, and 5
µg/ml pepstatin), and centrifuged at 10,000 x g for
60 min. For the NO synthase assay, macrophage lysates were incubated
with 2 mM L-arginine, 2 mM NADPH, 4 µM flavin-adenine
dinucleotide, 4 µM tetrahydrobiopterin, 2 mM DTT, and 1 mM EGTA for
2 h at 37°C. The reaction was terminated by adding 15 U/ml
L-lactic dehydrogenase and 83 mM sodium pyruvate and
incubating for an additional 15 min. Nitrite concentration in the
reaction mixture was measured as described above.
In addition, iNOS activity was also evaluated by measuring arginine to
citrulline conversion as previously described (22). Briefly, macrophage
monolayers were cultured in 12-well plates (4 x 106
cells/well in 2 ml of complete RPMI medium) in the presence of
LPS/IFN- and various concentrations of VIP or PACAP for 24 h.
The cells were washed twice in medium, and [3H]arginine
(3 µCi/ml) (Amersham, Arlington Heights, IL) was added for 20 min.
Incorporation of radiolabeled arginine was terminated by rapid
aspiration of the extracellular medium and two washes with ice-cold
PBS. The cells were lysed by the addition of 1 ml of 0.4 N
HClO4, and the intracellular extract was neutralized with
K2CO3 and 50 mM Tris/HCl buffer, pH 7.4.
Aliquots (100 µl) of the neutralized intracellular extract were used
to measure [3H]arginine uptake. To separate
[3H]citrulline from [3H]arginine, extract
aliquots of 400 µl were applied to 1-ml Dowex AG50WX-8
(Na+ form) columns as previously described (23). The
columns were eluted with three volumes of distilled water, and the
radioactivity in the flow-through fractions (containing almost
exclusively [3H]citrulline) was quantitated by
scintillation spectroscopy. All measurements were made in duplicate,
corrected for the number of counts per 4 x 106 cells,
and the data are presented as recovery of radiolabeled citrulline.
Western blot analysis
iNOS. Macrophage monolayers were washed three times with warm (37°C) PBS, harvested in cold 40 mM Tris/HCl buffer, pH 8, containing 5 µg/ml aprotinin, 1 µg/ml chymostatin, 50 µg/ml leupeptin, 5 µg/ml pepstatin, and 100 µM PMSF, and lysed by sonication. For detection of iNOS by Western blotting, 25 µg of macrophage lysates were separated on 7.5% SDS-PAGE gels under reducing conditions, transferred to reinforced nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany), and immunoblotted with anti-mouse iNOS mAb (Transduction Labs, Lexington, KY) as previously described (24).
NF-B and IRF-1.
Nuclear extracts were prepared from 1 x 107 cells by
resuspending the cells in 400 µl of ice-cold 10 mM HEPES, pH 7.9,
containing 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 sodium azide. After storage at 4°C for 10 min, Nonidet P-40 was
added to a final concentration of 0.5%, the cells were vortexed gently
for 15 s and centrifuged for 40 s at 12,000 x
g. The pelleted nuclei were washed once with the above
buffer and resuspended in 100 µl of ice-cold 20 mM HEPES, pH 7.9,
containing 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 sodium azide. After incubation on ice for
30 min with vigorous vortexing every 10 min, the nuclear proteins were
collected by centrifugation for 10 min at 12,000 x g.
For detection of NF-B and IRF-1, 25 µg of nuclear proteins were
separated on 7.5% SDS-PAGE gels under reducing conditions, transferred
to nitrocellulose membranes, and immunoblotted with Abs against p50,
p65, and IRF-1 (Santa Cruz Biotechnology) as recommended by the
manufacturer.
cAMP determinations
Following incubations for various times (from 0 to 120 min) with different concentrations of VIP, PACAP, or forskolin, macrophage monolayers (2 x 106 cells) were rinsed with ice-cold PBS and snap frozen in a dry ice-acetone bath. The cells were harvested in 1 ml of ice-cold 75% ethanol containing 0.5 mM 3-isobutyl-1-methylxanthine and centrifuged to remove the insoluble material. The supernatants were lyophilized and redissolved in 1 ml of 50 mM sodium acetate buffer, pH 6.2. The amounts of cAMP were quantitated by using a commercially available ELISA kit (Amersham).
RNA extraction and Northern blot analysis
Northern blot analysis was performed according to standard
methods. Macrophage monolayers (2 x 106 cells/ml)
were stimulated with LPS (0.5 µg/ml) and IFN- (200
U/ml), in the absence or 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 S&S Nytran membranes
(Schleicher and Schuell, Keene, NJ), and cross-linked to the nylon
membrane using UV light.
The probes for murine iNOS and ß-actin were generated by RT-PCR
as previously described (25, 26). 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 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 (Kodak,
Rochester, NY), and analyzed by densitometric analysis.
In vivo quantitation of NO production and iNOS activity and expression
Male mice (6–10 wk old) were injected with a single dose of LPS (100 µg/mouse) i.p. in the presence or absence of different amounts of VIP or PACAP (0.5–10 nmol/mouse). At different time points (2–8 h), blood was removed through cardiac puncture, and peritoneal exudate was obtained as described above. The peritoneal suspensions were centrifuged for 5 min at 1800 x g, and cell-free supernatants were harvested. Serum and peritoneal cell-free supernatants were assayed for NO production. The peritoneal cells were subjected to Northern blot analyses and assayed for iNOS activity as described above.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared by the miniextraction procedure of Schreiber et al. (27) with slight modifications. Raw 264.7 cells 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 final 0.5% 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. 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 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, 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 for later use in EMSA.
Oligonucleotides corresponding to the B (nucleotides -92 to -
65) and IRF-1 (nucleotides -933 to -906) motifs of the iNOS promoter
were synthesized (28, 29): 5'-CCAACTGGGGACTCTCCCTTTGGGAACA-3' (B),
and 5'-CACTGTCAATATTTCACTTTCATAATG-3' (IRF-1). The oligonucleotides
were annealed after 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. Aliquots of
50 ng of the double-stranded oligonucleotides were end-labeled with
[32P]ATP by using T4 polynucleotide kinase. For EMSA
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) were set up
containing 0.5–1 ng DNA probe, 5 µg nuclear extract, 2 µg
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
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.
Statistical analysis
All values are expressed as the mean ± SD of the number of experiments. Each sample was assayed in duplicate. Comparisons between groups were made using the 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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To determine the effect of VIP and PACAP on NO production, we
first determined the amounts of NO following treatment of
thioglycollate-elicited peritoneal macrophages with different stimuli.
Macrophages were stimulated with LPS, IFN-, or LPS plus IFN-.
Supernatants harvested 24 h later were assayed for the generation
of nitrite, an accumulated oxidative product of NO. Unstimulated
macrophages produce very low amounts of NO (Fig. 1
A). Detectable levels of NO
were present in macrophage cultures stimulated with all stimuli used,
with the highest NO production observed upon stimulation with LPS plus
IFN- (Fig. 1A). VIP and PACAP inhibited NO production by
95% in macrophages stimulated with LPS, and by 70% and 50% in
macrophages activated with IFN- or LPS plus IFN-, respectively
(Fig. 1A). In addition, VIP and PACAP inhibited in a dose-
and time-dependent manner the NO production by LPS- and
LPS/IFN--stimulated cells (Fig. 1, B and C).
The dose-response curves were similar for VIP and PACAP, showing
maximal effects at 10-8 M and an IC50 (i.e.,
the concentration of neuropeptide producing 50% of maximal inhibition)
of 0.50 nM (Fig. 1) for both stimuli. The time curves indicate that
the NO generation was significantly inhibited by VIP and PACAP as early
as 16 h, with the maximum inhibitory effect after 24 h of
culture (Fig. 1). Moreover, the reduction of NO generation was
maintained throughout the 72-h incubation period (data not shown),
indicating that VIP/PACAP do not delay, but rather reduce NO release.
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Optimal conditions were selected to further study the inhibitory effect
of VIP and PACAP on NO production, i.e., macrophages were stimulated
with LPS or LPS plus IFN- in the presence of 10-8 M
neuropeptide for 24 h.
VPAC1, and to a lesser degree VPAC2, mediate the inhibition of NO production by VIP and PACAP
Next we investigated whether the inhibitory effect of
VIP/PACAP could be related to 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. NO production was
not affected by secretin and glucagon (Fig. 2A). The two VIP fragments and
PACAP6–38 failed to inhibit NO generation, suggesting that
intact VIP and PACAP molecules are required for their inhibitory
activity (Fig. 2A).
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-induced NO production. VPAC1, and to a lesser degree VPAC2
agonists, but not the PAC1 agonist, inhibit NO release (Fig. 2
B). The VPAC1 agonist exhibits a potency similar to that of
VIP/PACAP (60% inhibition), whereas Ro 25-1553 was much less efficient
(17–24% inhibition) (Fig. 2B). In addition, we
investigated the ability of PACAP6–38, an antagonist
specific for PAC1 and, to a lesser degree for VPAC2 (37), and of a
specific VPAC1 antagonist (38), to reverse the effects of VIP and
PACAP. Increasing concentrations of the antagonists (10-8
to 10-6 M) were added simultaneously with a fixed
concentration of VIP or PACAP (10-8 M). The inhibitory
effects of VIP and PACAP were reversed by the VPAC1 antagonist in a
dose-dependent manner (Fig. 2C). In contrast,
PACAP6–38 did not reverse the inhibitory effect of VIP or
PACAP (Fig. 2D). Neither the VPAC1 antagonist nor
PACAP6–38 significantly affected NO levels (Fig. 2, C and D). Furthermore, the simultaneous addition
of VIP or PACAP and the VPAC1 agonist did not result in an additive
effect on NO generation (Fig. 2E). Together these results
confirm the specificity of the VIP and PACAP inhibitory activity, and
suggest that both neuropeptides exert their action primarily through
binding to VPAC1. Because VPAC2 appears to be expressed relatively late
following LPS stimulation (12 and 24 h) (see Footnote 4), the
lower efficiency of the VPAC2 agonist may result from a low number of
VPAC2 receptors early during culture. Effects of delayed addition of VIP and PACAP after macrophage activation
In the experiments described so far, VIP and PACAP were added to
cells at the same time as LPS. To find the level of VIP/PACAP-induced
blocking in NO generation, we next investigated the effect of exposing
macrophages to VIP or PACAP after LPS or IFN- stimulation. We
stimulated peritoneal macrophages with LPS (500 ng/ml) or
with IFN- (200 U/ml) and added 10-8 M VIP or PACAP at
different times after (from 0 to 3 h) the initiation of the
cultures. Supernatants were collected 24 h after the initiation of
the cultures and were assayed for NO production. The addition of VIP
and PACAP up to 2 h after LPS or IFN- stimulation resulted in
significant levels of inhibition (50% for LPS stimulation and 32% for
IFN- stimulation) (Fig. 3). Later
additions resulted in progressively lower degrees of inhibition (Fig. 3). Thus, VIP and PACAP block an early event in expression NO
generation activity in macrophages.
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Because all three types of VIP/PACAP receptors induce cAMP in
various cell types, and cAMP-inducing agents inhibit NO production
(39), we measured first the amounts of cAMP generated in
LPS/IFN--stimulated peritoneal macrophages after incubation with VIP
or PACAP. VIP and PACAP increased, in a dose-dependent and
time-dependent manner, the levels of intracellular cAMP (Fig. 4, A and B).
However, the effect of the two neuropeptides was lower than that of
forskolin (Fig. 4A).
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-stimulated macrophages, although
they show less of an effect at lower concentrations (10-8
and 10-7 M) as compared with VIP and PACAP (Fig. 5A). In addition, the
involvement of cAMP is supported by the results obtained with the two
protein kinase inhibitors. In contrast to calphostin C, H89 partially
reverses the inhibitory effect of VIP/PACAP (Fig. 5B). These
results suggest that the inhibitory effect of VIP/PACAP is mediated, at
least partially, through increases in intracellular cAMP.
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Concentrations of VIP and PACAP shown to efficiently suppress the
release of NO also reduce the expression of iNOS protein in
LPS/IFN--stimulated macrophages (Fig. 6A). The reduction in iNOS is
comparable in total peritoneal macrophage lysates, membrane and cytosol
preparations (data not shown). The reduction in iNOS protein expression
was paralleled by a decrease in iNOS enzymatic activity, as measured by
conversion of arginine to citrulline (Fig. 6B). VIP/PACAP
also decreased iNOS activity in macrophage lysates supplemented with
L-arginine and all cosubstrates and cofactors (data not
shown). Therefore, the reduced accumulation of nitrite in
VIP/PACAP-treated macrophage cultures reflects the decrease in iNOS
protein, and is not a consequence of L-arginine or
cosubstrate/cofactor depletion.
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Having demonstrated that VIP and PACAP inhibit NO production, we
sought to determine whether this action occurs at a transcriptional
level through the inhibition of iNOS mRNA expression. We stimulated
peritoneal macrophages with LPS plus IFN- in the presence or absence
of 10-8 M VIP or PACAP for 4, 8, 16, and 24 h, and
total RNA was prepared and subjected to Northern blot analysis.
Although no iNOS mRNA is detectable in unstimulated cells (Fig. 7), progressively increased levels of
iNOS mRNA are present in LPS/IFN--stimulated cells (up to 16 h)
(Fig. 7). At all time points, VIP and PACAP significantly inhibited the
levels of iNOS mRNA (Fig. 7), with a maximum effect at 16 h. The
amount of iNOS mRNA correlated with the generation of nitrite in
replica dishes (not shown). These results indicate that both
neuropeptides inhibit iNOS steady-state mRNA levels.
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B and IRF-1 binding
Although the iNOS promoter contains a complex array of
transactivating binding sites, NF-B and IRF-1 appear to be essential
for maximal iNOS transcription after LPS/IFN- stimulation (28, 29, 40). To investigate whether VIP/PACAP affect NF-B and IRF-1 binding,
we used the murine macrophage cell line Raw 264.7. First, we confirmed
that VIP and PACAP affect NO production in the Raw cells similar to
peritoneal macrophages. Indeed, VIP and PACAP inhibit, in a dose- and
time-dependent manner, the NO production in LPS/IFN--stimulated Raw
cells (Fig. 8A), with very
similar kinetics to those observed in peritoneal macrophages. In
addition, the two neuropeptides significantly reduce both iNOS activity
and expression in Raw cells (Fig. 8A). The effects of VIP
and PACAP on NF-B and IRF-1 were studied by EMSA. Stimulation of Raw
cells with LPS plus IFN- led to a time-dependent increase in both
NF-B and IRF-1 binding compared with unstimulated cells; treatment
with VIP and PACAP significantly inhibited the binding (Fig. 8, B and C, upper panels). The
specificity of the NF-B and IRF-1 binding was evident by the
complete displacement of the NF-B and IRF-1/DNA binding complexes in
the presence of a 50-fold excess of unlabeled homologous
oligonucleotides in the competition reactions (Fig. 8, B and
C, upper panels). In contrast, a 50-fold excess
of unlabeled nonhomologous oligonucleotides had no effects on this DNA
binding activity (Fig. 8, B and C, upper
panels). Ab supershift experiments were performed to determine the
composition of the NF-B and IRF-1-binding factors. Addition of
either monospecific anti-p50 or anti-p65 Abs to the binding
reaction resulted in a marked reduction in the intensity of the NF-B
band and led to the appearance of slow migrating bands, indicating that
the NF-B-binding complex is composed primarily of p50/p65
heterodimers (Fig. 8B, lower panel). Furthermore,
the IRF-1/DNA complexes were supershifted by an anti-IRF-1 Ab in
both LPS/IFN-- and VIP/PACAP-treated cells (Fig. 8C,
lower panel).
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B and
IRF-1 binding
Because the inhibitory effect of VIP on NO 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 and IRF-1 binding complexes. The inhibitory activity of VIP on
LPS/IFN--mediated NF-B binding was completely reversed by the
VPAC1 antagonist (Fig. 9A,
second panel, lane 3), but not by H89 (Fig. 9A, second panel, lane 4). However,
both the VPAC1 antagonist and H89 reversed the VIP inhibition of IRF-1
binding (Fig. 9A, first panel, lanes 3
and 4). These results suggest that the inhibition of NF-B
and IRF-1 binding by VIP are mediated through VPAC1, but only the
inhibition of the IRF-1 binding complex is entirely cAMP-dependent.
This is supported by the fact that forskolin (a cAMP inducer) does not
affect NF-B binding (Fig. 9A, second panel,
lane 5) but inhibits IRF-1 binding similar to VIP (Fig. 9A, first panel, lane 5 compared with
lane 1).
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B and
IRF-1 proteins, we performed Western blots (Fig. 9B). High
levels of p50, p65, and IRF-1 were present in nuclear extracts obtained
from Raw 264.7 cells stimulated with LPS/IFN- (Fig. 9B,
lane 1). In cells treated with VIP, the p50 levels remained
constant, but both p65 and IRF-1 became undetectable (Fig. 9B, lane 2). The VPAC1 antagonist restored both
p65 and IRF-1 levels (Fig. 9B, lane 3), whereas
the PKA inhibitor H89 restored only the IRF-1 levels (Fig. 9B, lane 4). These results indicate that the
reductions in the levels of p65 and IRF-1 nuclear proteins are mediated
through the VPAC1 receptor and confirm the involvement of cAMP in the
inhibitory effect of VIP on IRF-1, but not on NF-B. VIP and PACAP inhibit NO production and iNOS expression in endotoxic mice
TNF-, IL-1, IL-6, IFN-, IL-12, and more recently NO have
been shown to play pivotal roles in LPS-induced endotoxic shock (5, 6, 41, 42, 43). Indeed, inhibition of the LPS-induced cascade of
proinflammatory cytokines is the primary mechanism through which
anti-inflammatory immunomodulators such as IL-10, IL-11, and IL-13
confer protection against the lethal effects of LPS administration
(44, 45, 46, 47, 48). Based on the ability of VIP and PACAP to down-regulate
cytokine production by LPS-activated macrophages, we reasoned that the
in vivo protective effect of VIP/PACAP on endotoxin-induced lethal
septic shock (17) might be mediated by a similar mechanism. To evaluate
the effects of VIP and PACAP administration on NO production during
lethal endotoxemia, mice were injected i.p. with a LD90 of
LPS (100 µg) concurrently with either medium or different amounts of
VIP or PACAP (0.5–10 nmol). Serum and extracellular fluid
(peritoneal lavage) were collected at different time points, and NO
production was approximated from nitrite levels. Serum and peritoneal
nitrite levels peaked 4 h following LPS administration, and levels
were still relatively high at 8 h (Fig. 10A). In contrast, mice
receiving VIP or PACAP in combination with LPS showed a significant
reduction in serum and peritoneal NO levels (Fig. 10A). The
inhibitory effect was dose-dependent, with a maximum for 5–10 nmol
VIP/PACAP (Fig. 10A, lower panels). Consistent
with previous findings (49), serum and peritoneal TNF-, IL-6, IL-12,
and IFN- levels were significantly reduced by VIP and PACAP in these
samples (not shown). In addition, VIP and PACAP significantly inhibited
iNOS activity and transcription in peritoneal cells in endotoxic mice
2, 4, and 8 h after LPS challenge (Fig. 10, B and
C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The inhibitory effect is dose-dependent within a
wide range of neuropeptide concentrations (10-7 to
10-11 M), with the maximum effect being observed at
10-8 M. This is the dose range at which VIP and PACAP
modulate several immunological functions (11, 12).
Similar to the effect on cytokines such as IL-2, IL-6, IL-10, IL-12,
and TNF- (13, 16, 52, 53; Footnote 4), the inhibition of NO
production requires intact VIP/PACAP molecules. This is in agreement
with previous reports showing that both C- and N-terminal truncations
of VIP lead to significant losses in biological activity (54, 55).
Peritoneal macrophages and the Raw 264.7 macrophage cell line have been
shown to constitutively express VPAC1 and PAC1 mRNA and VPAC2 mRNA
following LPS-stimulation (31–33; Footnote 4). Our agonist studies
suggest that VPAC1 is the major mediator of the VIP/PACAP inhibitory
effect on NO generation. The VPAC2 agonist Ro 25-1553 was much less
efficient; however, because VPAC2 is expressed only later in activated
macrophages, the lack of effectiveness for the Ro compound may be due
to a lack of appropriate receptors during the early culture period. The
role of VPAC1 as the major player in mediating the effect of VIP/PACAP
on NO 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 (37), reverses the inhibitory effect of
VIP/PACAP. Also, the VPAC1 antagonist blocked the effect of VIP/PACAP
on IRF-1 and NFB binding to the iNOS promoter, supporting the
involvement of the VPAC1 in the inhibition of iNOS expression.
The VPAC1 is coupled primarily to the adenylate cyclase system (56),
and NO production is indeed inhibited by agents that increase
intracellular cAMP levels (39). In the present study, VIP and PACAP
induced intracellular cAMP in a dose-dependent manner, and forskolin
and PGE2, two strict cAMP-inducing agents, inhibited NO
generation. 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 previously made for the
inhibitory effect of VIP/PACAP on TNF- and IL-12 production in
macrophages (33; Footnote 4) and on IL-2 and IL-10 production in
lymphocytes (our unpublished observations). The existence of a
second cAMP-independent pathway is also supported by the fact that at
concentrations that are physiologically relevant for VIP
(10-9 M), the peptide induced less cAMP than forskolin,
while acting as a more potent NO inhibitor. The nature of this second
transduction pathway remains to be determined.
The iNOS activity is regulated at various levels, such as
transcriptional, posttranscriptional, translational, and
posttranslational. However, iNOS transcription appears to be the
primary regulatory site (57). The present study indicates that the
inhibitory effect of VIP and PACAP on NO production occurs through the
reduction in iNOS protein and mRNA levels. The precise molecular
mechanisms that account for the VIP/PACAP inhibition of iNOS expression
are largely unknown, and it remains to be established whether the
reduction in steady-state iNOS mRNA levels results from a decrease in
de novo transcriptional rate, message stabilization, or both. However,
some evidence points to a direct effect of VIP/PACAP on the de novo
transcription as the most likely possibility. Delayed treatment with
VIP/PACAP did not affect NO production, suggesting that VIP and PACAP
inhibit an early event in iNOS expression. The promoter of the iNOS
gene contains two major discrete regions that synergize upon binding of
transcriptional factors (28, 29, 40, 58, 59): a NF-B binding site,
activated mainly by LPS, and an IRF-1 binding site, for IFN--induced
transcriptional factors. As VIP and PACAP inhibit NO production induced
by LPS and IFN-, the two neuropeptides might regulate the activation
of both transactivating sites. The present study indicates that VIP and
PACAP inhibit NF-B (p50/p65) binding and nuclear p65 protein levels
in LPS/IFN--stimulated Raw 264.7 cells. We have recently reported a
similar effect for the B site specific for the TNF- promoter
(33). It remains to be determined whether the VIP/PACAP reduction in
NF-B binding and nuclear p65 levels are due to an increase in IB
protein levels, a decrease in IB degradation, or/and inhibition in
IB phosphorylation, as described for other anti-inflammatory
agents, such as IL-11, IL-10, TGF-ß1, glucocorticoids, and
antioxidants (44, 60, 61, 62, 63).
In addition, treatment of the Raw 264.7 cells with LPS plus IFN-
resulted in a marked IRF-1 binding that was strongly inhibited by VIP
or PACAP. Similar to p65, the levels of nuclear IRF-1 protein were
reduced by VIP. Although the precise mechanism underlying this effect
remains to be determined, this finding points to a VIP/PACAP-sensitive
step in the activation of IRF-1, which unlike NF-B is not
constitutively present in the cytosol in an inactive form, but
synthesized de novo following exposure to IFN- (64). In macrophages,
the IRF-1 gene responds to IFN-, through binding of the IFN-gamma
activation factor complex generated by the Jak1/2-STAT1 pathway
(reviewed in 65). Could VIP and PACAP affect the Jak-STAT
signaling? There is indeed evidence that cAMP-elevating agents inhibit
the activation of the STAT1 pathway (66, 67, 68). We investigated the
relationship between the cAMP/PKA pathway and the IRF-1 binding
activity. Both VIP and forskolin inhibit IRF-1 binding, and H89
reverses the effect of VIP on IRF-1 binding. The inhibition of IRF-1
binding could be due to a reduction in IRF-1 expression, most probably
by down-regulating STAT1 activation. In contrast to IRF-1, increases in
cAMP do not appear to directly affect NF-B binding. Forskolin does
not affect NF-B binding, and H89 does not reverse the inhibitory
effect of VIP. Similar results were obtained for the NF-B binding to
the TNF- promoter in LPS-stimulated macrophages (33). We propose
that, similar to TNF- transcription, the B transactivation event
required for iNOS expression is mediated through the cAMP-independent
transduction pathway.
Production of NO by iNOS is beneficial for the protection against
bacteria, fungi, parasites, viruses, and tumor cells (1, 4), but its
overproduction can be harmful in endotoxemia, neurologic disorders,
rheumatoid arthritis, and autoimmune diseases (5, 6, 7). Inhibitors of all
NOS isoforms, which have been used to treat some of these diseases, led
to significant harmful side-effects, such as hepatic injury, and even
increased mortality (69, 70). Also, the clinical use of the NOS
inhibitors has been hampered by the hypertensive effect resulting from
the nonspecific inhibition of the constitutive NOS isoforms. Therefore,
a specific iNOS inhibitor, with little or no effect on nNOS, holds
significant therapeutical potential. The iNOS specificity of VIP
initially documented by Bandyopadhyay et al. in rat (51) has to be
confirmed in other species, and the molecular mechanisms allowing the
differential regulation of iNOS and nNOS have to be clarified. However,
based on their ability to down-regulate iNOS expression and NO
production in endotoxemic mice, as described in this study, VIP/PACAP
are attractive candidates for the development of treatments for septic
shock and other acute inflammatory diseases, as well as for autoimmune
diseases. In fact, VIP and PACAP protect mice from endotoxic shock even
when administered post-LPS (17). Unlike neutralizing Abs and receptor
antagonists directed against a single cytokine, VIP and PACAP reduce
the production of a wide spectrum of proinflammatory mediators. In
vitro and in vivo studies indicate that VIP and PACAP directly interact
with macrophages to suppress the LPS-induced production of TNF-,
IL-6, IFN-, IL-12, and NO (13, 16, 33).
In conclusion, we have shown that the binding of VIP and PACAP,
primarily to VPAC1, inhibits iNOS expression at a transcriptional level
in LPS/IFN--stimulated macrophages through two intracellular
pathways: a cAMP-dependent pathway that preferentially inhibits IRF-1
transactivation and a cAMP-independent pathway that blocks NF-B
binding to the iNOS promoter. The inhibition of iNOS transcription by
VIP/PACAP may have therapeutical potential, because excessive NO
production has been implicated in the tissue injuries characteristic
for several inflammatory and autoimmune diseases.
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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: NO, nitric oxide; IRF-1, IFN regulatory factor-1; EMSA, electrophoretic mobility shift assay; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; PAC1, pituitary adenylate cyclase-activating polypeptide receptor; PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A; VIP, vasoactive intestinal peptide; VPAC1, type 1 vasoactive intestinal peptide receptor; VPAC2, type 2 vasoactive intestinal peptide receptor.
Received for publication August 19, 1998. Accepted for publication January 19, 1999.
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