J Biol Chem, Vol. 273, Issue 47, 31427-31436, November 20, 1998
Vasoactive Intestinal Peptide and Pituitary Adenylate
Cyclase-activating Polypeptide Inhibit Tumor Necrosis Factor 
Transcriptional Activation by Regulating Nuclear Factor-kB and cAMP
Response Element-binding Protein/c-Jun*
Mario
Delgado
§,
Ernesto J.
Munoz-Elias,
Yanqing
Kan,
Illana
Gozes¶,
Mati
Fridkin
,
Douglas E.
Brenneman**,
Rosa P.
Gomariz§, and
Doina
Ganea
From the Department of Biological Sciences, Rutgers
University, Newark, New Jersey 07102, § Departamento
Biologia Celular, Universidad Complutense, Madrid 28040, Spain,
¶ Department of Clinical Biochemistry, Sackler School of Medicine,
Tel Aviv University, Tel Aviv 69978, Israel, Department of
Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel, and ** Section on Developmental and Molecular Pharmacology,
Laboratory of Developmental Neurobiology, NICHD, National Institutes of
Health, Bethesda, Maryland 20895
 |
ABSTRACT |
Tumor necrosis factor 
(TNF), an early
cytokine produced by activated macrophages, plays an essential role in
normal and pathological inflammatory reactions. The excessive
production of TNF is prevented by the so-called
"macrophage-deactivating factors." This study examines the role of
two structurally related neuropeptides, the vasoactive intestinal
peptide (VIP) and the pituitary adenylate cyclase-activating peptide
(PACAP), as inhibitors of TNF. Both VIP and PACAP inhibit TNF
production from lipopolysaccharide-stimulated RAW 246.7 cells in a
dose- and time-dependent manner. Although the activated
cells express mRNA for all three VIP/PACAP receptors, agonist and
antagonist studies indicate that the major receptor involved is
VIP1R. VIP/PACAP inhibit TNF gene expression by
affecting both NF-kB binding and the composition of the cAMP responsive element binding complex (CREB/c-Jun). Two transduction pathways, a
cAMP-dependent and a cAMP-independent pathway, are involved in the inhibition of TNF gene expression and appear to
differentially regulate the transcriptional factors involved. Because
TNF plays a central role in various inflammatory diseases such as
endotoxic shock, multiple sclerosis, cerebral malaria, and various
autoimmune conditions, the down-regulatory effect of VIP/PACAP may have
a significant therapeutic potential.
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INTRODUCTION |
Macrophages are widely recognized as cells that play a central
role in the regulation of immune and inflammatory activities, as well
as tissue remodeling. The execution of these activities is mediated by
complex and multifactorial processes involving macrophage products (1).
In response to antigens such as
LPS,1 macrophages secrete
proinflammatory cytokines and oxidants such as TNF, IL-6, IL-1
,
IL-12, and nitric oxide (1). TNF and IL-6 are important macrophage
secretory products that contribute to pathophysiological changes
associated with several acute and chronic inflammatory conditions,
including septic shock, autoimmune diseases, wasting, rheumatoid
arthritis, inflammatory bowel disease, and respiratory distress
syndrome (2-4). A number of regulatory molecules termed
macrophage-deactivating factors have been the focus of considerable
research (5-9). These molecules are believed to prevent the excessive
production of proinflammatory mediators, including TNF and IL-6.
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 inhibit several macrophage
functions, including phagocytosis, respiratory burst, and chemotaxis
(reviewed in Ref. 10), as well as LPS-induced IL-6 production (11). Furthermore, we have recently demonstrated that VIP and PACAP protect
mice from endotoxic shock presumably through the inhibition of TNF
and IL-6 production.2 VIP was
also reported to suppress TNF production in human peripheral blood
cells (12, 13).
Both VIP and PACAP interact with a family of three VIP/PACAP receptors,
VIP1R, and VIP2R, which exhibit similar
affinities for the two neuropeptides and activate primarily the
adenylate cyclase system, and PACAP-R, which exhibits a 2-3 orders of
magnitude higher affinity for PACAP than for VIP and activates both the adenylate cyclase and phospholipase C systems (reviewed in Ref. 14).
Peritoneal macrophages have been described to possess VIP1R and PACAP-R (15-17).
LPS is a major stimulus for the production of proinflammatory
cytokines, including TNF, from macrophages (1). TNF
synthesis is controlled at several levels. Whereas
post-transcriptional, translational, and post-translational mechanisms
play important roles, TNF transcription appears to be the primary
regulatory site. Although the TNF promoter contains a complex array
of transactivating binding sites, the kB and CRE elements appear
essential for maximal TNF transcription (18-21).
To further understand the molecular mechanism through which VIP and
PACAP attenuate the inflammatory response, we have examined the effects
of both neuropeptides on TNF protein and mRNA levels in
LPS-activated Raw 264.7 macrophages and sought the specific receptor,
the intracellular signal pathway, as well as the possible nuclear
factors involved.
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EXPERIMENTAL PROCEDURES |
Reagents--
Synthetic VIP, PACAP38, VIP1-12, and
VIP10-28 were purchased from Novabiochem (Laufelfingen,
Switzerland). The VIP1R-antagonist
(Ac-His1,D-Phe2,Lys15,Arg16,Leu27)VIP(3-7)-GRF(8-27)
and the VIP1R-agonist
(Lys15,Arg16,Leu27)VIP(1-7)-GRF(8-27)
were kindly donated by Dr. Patrick Robberecht (Universite Libre de
Bruxelles, Belgium). The VIP2R-agonists Ro 25-1392 Ac-(Glu8,O-CH3-Tyr10,Lys12,Nle17,Ala19,Asp25,Leu26,Lys27,28)-VIP
cyclo(21-25) and Ro 25-1553
Ac-(Glu8,Lys12,Nle17,Ala19,Asp25,Leu26,Lys27,28,Gly29,30,Thr31)-VIP
cyclo(21-25) were generous gifts from Drs. Ann Welton and David R. Bolin (Hoffmann-La Roche). The PACAP-R agonist maxadilan was a generous
gift from Dr. Ethan A. Lerner (Massachusetts General Hospital,
Charlestown, MA). The lipophilic VIP agonist
stearyl-norleucine17 VIP (SNV) and antagonist
stearyl-norleucine17 neurotensin-VIP hybrid (SANV) were
previously described (22). The PACAP-R-antagonist
PACAP6-38, secretin, and glucagon were obtained from
Peninsula Laboratories (Belmont, CA). LPS (from E. coli
055:B5), calphostin C, and forskolin were purchased from Sigma, and
N-[2-(p-bromocinnamyl-amino)ethyl]-5-iso-quinolinesulfonamide (H89) from ICN Pharmaceuticals, Inc. (Costa Mesa, CA). Antibodies against CREB, p50, p65, and c-Jun were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cell Lines and Cell Culture--
The murine macrophage cell line
Raw 264.7 (ATCC, Manassas, VA) was grown as recommended by ATCC. The
cells were seeded in flat bottom 96-well microtiter plates (Corning
Glass, Corning, NY) at 8 × 104 cells/well in a final
volume of 200 µl. 24 h later the monolayers were washed twice in
Dulbecco's modified Eagle's medium without serum, and 200 µl of
10% Dulbecco's modified Eagle's medium was added to each well. The
cells were stimulated with LPS in the presence or absence of VIP or
PACAP38. Cell-free supernatants were harvested at designated time
points and assayed for TNF production by ELISA.
Cytokine Determination: ELISA Assay for TNF--
The amount
of soluble TNF was determined by using a murine TNF ELISA assay
with capture Ab (clone MP6-XT22) and detection Ab (biotinylated clone
MP6-XT3) with murine TNF (Pharmingen, San Diego, CA) as standard.
The ELISA is specific for murine TNF (does not cross-react with
human or rat TNF, or with other murine cytokines such as IL-10,
IL-6, IL-2, IL-3, IL-4, IL-5, IL-1, IL-1, and interferon-
).
The sensitivity of the assay is 10 pg TNF/ml.
RNA Isolation and Northern Blot Analysis--
Northern blot
analysis was performed according to standard methods. The cells (2 × 106 cells/ml) were stimulated with LPS (1 µg/ml) in
the absence or presence of VIP or PACAP38 (10
8
M) for different time periods, and total RNA was isolated
with Ultraspec RNA reagent (Biotecx Labs, Houston, TX) as recommended by the manufacturer. 20 µg of total RNA were electrophoresed on 1.2%
agarose-formaldehyde gels, transferred to S&S Nytran membranes (Schleicher & Schuell), and UV cross-linked. The TNF probe
oligonucleotide 5'-TTGACCTCAGCGCTGAGTTGGTCCCCCTTCTCAGCTGGAAGACT-3' was
designed based on the published murine TNF sequence (23). The
following murine 18 S rRNA oligonucleotide
5'-CCAAGGACAGGGCCTCGAAAGAGTCCTGTA-3' was used as control. The membranes
were prehybridized for 16 h at 42 °C, followed by hybridization
at 60 °C for 16 h with the appropriate probes. The
prehybridization and hybridization buffers were purchased from 5 Prime
3 Prime, Inc. (Boulder, CO).
Reverse Transcription-PCR for the Detection of VIP1R,
VIP2R, and PACAP-R mRNA--
2 µg of total RNA from
unstimulated and LPS-stimulated Raw 264.7 cells was reverse transcribed
and 3 µl of cDNA was amplified with specific primers. The primers
for VIP2R and PACAP-R were designed based on published
murine pancreatic VIP2R (24) and mouse brain PACAP-R (25),
respectively. The primers for VIP1R were previously
described (26, 27). The designated primers sequences are as follows:
VIP1R sense, 5'-CCTTCTTCTCTGAGCGGAAGTACTT-3' and antisense,
5'-CCTGCACCTCACCATTGAGGAAGCAG-3'; VIP2R sense, 5'-GTCAAGGACAGCGTGCTCTACTCC-3' and antisense,
5'-CCCTGGAAGGAACCAACACATAAG-3'; PACAP-R sense, 5'-CAAG
AAGGAGCAAGCCATGTGC-3' and antisense, 5'-CATCGAGTAATGGGGGAAGGG-3'; -actin sense, 5'-GATGGTGGGTATGGGTCAGGGG-3' and antisense,
5'-GCTCATTGCCGATAGTGATGACCT-3'. The expected sizes for the amplified
fragments are: 450 bp for VIP1R, 572 bp for
VIP2R, 317 bp for PACAP-R, and 660 bp for -actin. The
PCR conditions were: denaturation 94 °C, 45 s, annealing
55 °C, 45 s, primer extension 72 °C, 90 s for 35 cycles. The PCR products were size separated in 2% agarose gels.
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared by the mini-extraction procedure of Schreiber et
al. (28) with slight modifications. Raw 264.7 cells were plated at
a density of 107 cells/well in 6-well plates, stimulated
and washed twice with ice-cold phosphate-buffered saline and 0.1%
bovine serum albumin. The cell pellets were homogenized with 0.4 ml of
buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 5 mM NaF, 1 mM Na3VO4,
and 1 mM NaN3). After 15 min on ice, Nonidet
P-40 was added to a final 0.5% concentration, and nuclei were isolated
by centrifugation at 12,000 × g for 40 s.
Pelleted nuclei were washed once with 0.2 ml of ice-cold buffer A and
lysed by incubation for 30 min on ice in 0.1 ml of buffer C (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM
EDTA, 1 mM EGTA, 25% glycerol, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM NaN3). Supernatants containing nuclear proteins were harvested by centrifugation for 10 min at 14,000 rpm at
4 °C, the protein concentration was determined by the Bradford method, and aliquots were stored at 
80 °C for use in
electrophoretic mobility shift assays (EMSAs).
EMSA--
Oligonucleotides corresponding to the proximal (510
bp) kB3 (5'-CAAACAGGGGGCTTTCCCTCCTC-3') and CRE
(5'-TCCACATGAGATCATGGTTT-3') (106 bp) motif of the TNF
promoter (19, 23) were synthesized, annealed, and end-labeled with
[-32P]ATP. 20,000-50,000 cpm of double-stranded
oligonucleotide, corresponding to approximately 0.5 ng, was used for
each reaction. Binding reaction mixtures (15 µl) 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 dithiothreitol, 5%
glycerol, and 10 mM Tris-HCl, pH 7.5) were incubated on ice
for 15 min, followed by the addition of the probe. After 20 min of
incubation at room temperature, the 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), followed by transfer and autoradiography. In
competition and antibody supershift experiments, nuclear extracts were
incubated for 15 min at room temperature with antibody (1 µg) or
competing oligonucleotide (50-fold excess) before the addition of the
labeled probe.
JNK Activity Assay--
JNK activity was determined using a
stress-activated protein kinase/JNK assay kit (New England BioLabs,
Inc., Beverly, MA) according to the manufacturer's instructions.
Briefly, Raw 264.7 cell lysates, prepared according to the
manufacturer's instructions, were incubated with an N-terminal c-Jun
(1-89) fusion protein bound to glutathione-Sepharose beads to
selectively isolate JNK, followed by a kinase reaction in the presence
of cold ATP. Proteins were resolved on 12% SDS-polyacrylamide gel
electrophoresis gels, and electrophoretically transferred to
nitrocellulose membranes. c-Jun phosphorylation at Ser63
was selectively analyzed by immunoblotting using a phospho-specific c-Jun antibody and chemiluminescent detection.
Statistical Analysis--
All values are expressed as the
mean ± S.D. of the number of experiments performed in duplicate,
as indicated in the corresponding figures. 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 |
VIP and PACAP Inhibit LPS-induced TNF Production by Raw 264.7 Macrophages--
To investigate whether VIP/PACAP inhibit TNF
release, Raw 264.7 murine macrophages were stimulated with different
concentrations of LPS in the absence or presence of various doses of
VIP or PACAP, and the amount of TNF released in the culture
supernatants was assayed by ELISA at different time periods. VIP and
PACAP inhibit the TNF production by LPS-stimulated cells in a dose-
and time-dependent manner, showing maximal effects at
108 M after 6 h of incubation (Fig.
1).
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Fig. 1.
VIP and PACAP inhibit TNF production by
LPS-stimulated macrophages. A, Raw 264.7 cells were
stimulated with a concentration range of LPS (10 pg/ml to 10 µg/ml)
in the absence or presence of 108 M VIP or
PACAP. After a 6-h incubation period, supernatants were collected, and
TNF release was determined by ELISA. Control cultures were incubated
with LPS alone. B, time course for the inhibitory effect of
VIP/PACAP on TNF production. Raw 264.7 cells were stimulated with
LPS (1 µg/ml) in the absence or presence of 108
M VIP or PACAP. Supernatants collected at different times
were assayed for TNF production by ELISA. C,
dose-response curve for the inhibitory effect of VIP and PACAP on
TNF production. Raw 264.7 cells were incubated with LPS (1 µg/ml)
and a concentration range of either VIP or PACAP for 6 h.
Supernatants were collected and TNF release was determined by ELISA.
For A-C, cells cultured in the absence of LPS with or
without VIP/PACAP did not produce detectable levels of TNF (<10
pg/ml). Each result is the mean ± S.D. of five separate
experiments performed in duplicate. * p < 0.001 with
respect to control cultures with LPS alone.
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The Inhibition of LPS-induced TNF Production by VIP or PACAP Is
Mediated through VIP1R--
Next we investigated whether
the inhibitory effects of VIP/PACAP could be related to occupancy of
specific receptors. First, we compared the effect of VIP/PACAP to
secretin, glucagon, and the VIP- and PACAP-fragments
VIP1-12, VIP10-28, and PACAP6-38. TNF production was inhibited by secretin
only at 107 M, whereas no effect was observed
with various concentrations of glucagon
(109-107 M) (Fig.
2A). The two VIP fragments and
PACAP6-38 failed to inhibit TNF production, suggesting
that intact VIP and PACAP molecules are required for their inhibitory
activity (Fig. 2A).
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Fig. 2.
Inhibition of TNF production by VIP and
PACAP is specific and is mediated through VIP1R.
A and B, comparative effects of VIP, PACAP38,
VIP-related peptides, VIP fragments, and VIP and PACAP agonists on
TNF production. Raw 264.7 cells were stimulated with LPS (1 µg/ml)
in the absence or presence of different concentrations of secretin,
glucagon, VIP1-12, and VIP10-28
(A), or maxadilan (PACAP-R-agonist), Ro 25-1632, Ro 25-1553 (VIP2R-agonists), or
(Lys15,Arg16,Leu27)VIP(1-7)-GRF(8-27)
(VIP1R-agonist) (B). Supernatants were collected
6 h later and assayed for TNF production by ELISA. Each result
is the mean ± S.D. of four experiments performed in duplicate. *
p < 0.05, ** p < 0.01, and ***
p < 0.001 with respect to control cultures with LPS
alone. C and D, effect of PACAP-R and
VIP1R antagonists on the inhibitory activity of VIP and
PACAP on TNF production. Raw 264.7 cells were stimulated with LPS (1 µg/ml), and treated simultaneously with VIP or PACAP
(108 M), and different concentrations of the
VIP1R-antagonist
(Ac-His1,D-Phe2,Lys15,Arg16,Leu27)VIP(3-7)-GRF(8-27)
(C), or the PACAP-R antagonist (PACAP6-38) (D).
Supernatants were collected 6 h later and assayed for TNF.
VIP1R antagonist and PACAP6-38 did not affect TNF
levels (1577 ± 112 pg/ml for 106 M
VIP1R antagonist; 1516 ± 145 pg/ml for
106 M PACAP6-38 compared with 1562 ± 135 pg/ml for LPS alone). Percentage of inhibition (D) was
calculated by comparison with controls containing LPS alone. Each
result is the mean ± S.D. of four experiments performed in
duplicate. * p < 0.05, *** p < 0.001 compared with samples treated with neuropeptides and without
antagonists. E and F, effect of
VIP1R, VIP2R, and PACAP-R agonists. Raw 264.7 cells were stimulated with LPS (1 µg/ml) and treated with a
VIP1R agonist (100 nM) (E), or
maxadilan and/or Ro 25-1553 (100 nM) (F). VIP or
PACAP (108 M) were added at the same time or
15 min after the agonists (F). Supernatants were collected
6 h later and assayed for TNF production. Percentage of
inhibition (F) was calculated by comparison with controls
containing LPS alone. Results are the mean ± S.D. of five
experiments performed in duplicate. * p < 0.001 with
respect to samples treated with VIP.
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To determine which of the VIP/PACAP receptors were involved, we used
specific receptor agonists and antagonists. We investigated the effect
of a newly described VIP1R-agonist (29), of two
VIP2R agonists (Ro 25-1392 and Ro 25-1553) (30, 31), and of
maxadilan, a specific PACAP-R agonist (32) on the LPS-induced TNF
production. VIP1R, VIP2R, and PACAP-R agonists
inhibited TNF release (Fig. 2B). The VIP1R
agonist exhibited a similar potency as VIP/PACAP (60% inhibition),
whereas maxadilan and the two Ro compounds were much less efficient
(22-26% inhibition) (Fig. 2B). In addition, we
investigated the ability of PACAP6-38, an antagonist specific for PACAP-R and VIP2R (33), and of a specific
VIP1R-antagonist (34), to reverse the effects of VIP and
PACAP. Increasing concentrations of the antagonists
(106-108 M) were added
simultaneously with a fixed concentration of VIP or PACAP
(108 M). The inhibitory effects of VIP and
PACAP were reversed by the VIP1R-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 VIP1R-antagonist
nor PACAP6-38 significantly affected TNF levels (Fig.
2, C and D legends). Furthermore, the
simultaneous addition of VIP or PACAP and VIP1R-agonist did
not result in an additive effect on TNF release (Fig.
2E). Together these results confirm the specificity of the
VIP and PACAP inhibitory activity and suggest that both neuropeptides exert their action through binding to VIP1R.
Finally, the simultaneous addition of VIP with maxadilan and/or Ro
25-1553 did not result in significant differences in comparison with
samples treated with VIP alone; however, a small but statistically significant difference was observed when the cells were preincubated for 15 min with PACAP-R and/or VIP2R agonists before the
addition of VIP (Fig. 2F).
Raw 264.7 Cells Express VIP1R, VIP2R, and
PACAP-R--
The fact that VIP1R, VIP2R, and
PACAP-R agonists inhibit TNF production suggests that Raw 264.7 cells express all three receptors. To test this possibility, we
investigated the expression of VIP1R, VIP2R,
and PACAP-R mRNA by reverse transcription-PCR in unstimulated and
LPS-stimulated Raw 264.7 cells. Both VIP1R- and
PACAP-R-specific fragments were amplified from unstimulated and
stimulated macrophages, whereas VIP2R fragments were only
detected in stimulated cells (Fig. 3).
These results indicate that LPS-stimulated Raw 264.7 cells express
VIP1R, VIP2R, and PACAP-R mRNA.
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Fig. 3.
Expression of VIP1R,
VIP2R, and PACAP-R mRNA in Raw 264.7 cells. Total
RNA extracted from unstimulated macrophages (lane 2) and
LPS-stimulated macrophages (lane 3) (2 × 107 cells) was subjected to reverse transcription-PCR with
specific primers for VIP1R, VIP2R, PACAP-R, and
-actin as described under "Experimental Procedures." Brain RNA
was used as a positive control (lane 1). Reactions without
cDNA served as negative control (lane 4). Numbers
indicate the predicted sizes for the amplified fragments. One
representative experiment of two is shown.
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Intracellular Signal Pathways Involved in the Inhibitory Activity
of VIP and PACAP on TNF Production--
cAMP but not protein kinase
C involvement. To study the second messengers involved in the
inhibitory activity of VIP and PACAP, we investigated the effects of
calphostin C (a protein kinase C inhibitor) (35) and of H89 (a protein
kinase A inhibitor) (36) on the inhibition of TNF. High
concentrations (100 nM) of calphostin C inhibited TNF
production in LPS-treated cells (Fig.
4C); however, in the 1-10
nM concentration range, calphostin C did not affect TNF
production in LPS-stimulated cells and did not reverse the inhibitory
effect of VIP or PACAP (Fig. 4, A and B). In
contrast, H89 partially reversed the inhibitory effect of VIP and PACAP
(Fig. 4, A and B). These results suggest that the
inhibitory effect of VIP/PACAP is mediated, at least partially, through
increases in intracellular cAMP.
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Fig. 4.
Comparative effects of calphostin C (a
protein kinase C inhibitor) and H89 (protein kinase A inhibitor) on the
inhibitory activity of VIP and PACAP. Raw 264.7 cells were
stimulated with LPS (1 µg/ml), LPS plus VIP (108
M) (A), or LPS plus PACAP (108
M) (B) in the absence or presence of different
concentrations of calphostin C or H89. After a 6-h culture the
supernatants were assayed for TNF production. The dashed
horizontal line represents control values from cultures incubated
with LPS alone (1659 ± 141 pg TNF/ml). Results are the
mean ± S.D. of five experiments performed in duplicate. *
p < 0.001 with respect to neuropeptide-treated samples
without protein kinase modulators.  , calphostin C;  , H89.
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Involvement of a cAMP-independent Pathway--
The partial
reversal of the inhibitory effect of VIP on TNF production by H89
suggests the involvement of an additional cAMP-independent transduction
pathway. To address this question, we used a lipophilic VIP agonist
(SNV) and a lipophilic VIP antagonist (SANV) previously developed for
the neurotrophic action of VIP, which act through cAMP-independent
pathways (22). SNV inhibits TNF production, although less
efficiently than VIP or PACAP (Fig.
5A), and the inhibitory
activity is not reversed by H89 (Fig. 5B). This suggests the
existence of a second cAMP-independent pathway in the transduction of
the VIP signaling in macrophages. To further substantiate this possibility, we investigated whether SNV contributes to the inhibition of TNF by forskolin or prostaglandin E2
(PGE2), two strict cAMP-inducing agents. Although SNV did
not significantly affect the VIP/PACAP inhibitory effect, it increased
the inhibitory action of forskolin and PGE2 (Fig.
5C). The effect is additive, and not synergistic, suggesting
that the two pathways are probably not connected.
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Fig. 5.
Involvement of a cAMP-independent pathway in
the inhibitory effect of VIP/PACAP on TNF production.
A, effect of the lipophilic VIP agonist SNV. Raw 264.7 cells
were stimulated with LPS (1 µg/ml) and incubated with different
concentrations of SNV, VIP, or PACAP. Supernatants were collected
6 h later and assayed for TNF production. Each result is the
mean ± S.D. of four experiments performed in duplicate. *
p < 0.001 compared with controls with LPS alone.
B, effect of calphostin C and H89 on the inhibitory activity
of SNV. Raw 264.7 cells were stimulated with LPS (1 µg/ml) or LPS
plus SNV (107 M) in the absence or presence
of different concentrations of calphostin C or H89. Supernatants were
collected 6 h later and assayed for TNF production. The
dashed horizontal line represents control values from
cultures with LPS alone. C, effect of SNV on the inhibitory
action of VIP, PACAP, and other cAMP-elevating agents. Raw 264.7 cells
were stimulated with LPS (1 µg/ml) and incubated with VIP
(108 M), PACAP (108
M), FK (106 M), or
PGE2 (106 M) in the presence or
absence of 100 nM SNV. Supernatants were collected 6 h
later and assayed for TNF production. Percentage of inhibition was
calculated by comparison with LPS controls (1,690 ± 159 pg
TNF/ml). Numbers in parenthesis represent expected percentages of
inhibition if the effects of SNV and others agents were additive. Each
result is the mean ± S.D. of four experiments performed in
duplicate. D, effect of SANV, a lipophilic VIP antagonist,
on the inhibitory activity of VIP, FK, 8-Br-cAMP, and PGE2.
Raw 264.7 were stimulated with LPS (1 µg/ml) and incubated with VIP
(108 M), PACAP (108
M), FK (106 M), 8-Br-cAMP
(107 M), or PGE2
(106 M) in the presence or absence of
different concentrations of SANV. Supernatants were collected 6 h
later and assayed for TNF production. The dashed horizontal
line represents control values from cultures incubated with LPS
alone. Results are the mean ± S.D. of five experiments performed
in duplicate. * p < 0.001 with respect to samples
treated with VIP.
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In addition, we investigated the effect of the antagonist SANV on the
inhibition of TNF production by VIP, PACAP, forskolin, PGE2, and 8-Br-cAMP. SANV partially reverses the inhibitory
effect of VIP, PACAP, forskolin, PGE2, and 8-Br-cAMP (Fig.
5D), suggesting that SANV acts on a messenger downstream
from cAMP.
VIP and PACAP Regulate TNF Production at a Transcriptional
Level--
To determine whether the VIP/PACAP affect TNF
transcription, Raw 264.7 cells were stimulated with LPS in the presence
or absence of 108 M VIP or PACAP for 1.5, 3, and 6 h, and total RNA was prepared and subjected to Northern blot
analysis. Although no TNF mRNA is detectable in unstimulated
cells, time-dependent increasing levels of TNF mRNA
are present in LPS-stimulated cells (Fig. 6). At all three time points, VIP and
PACAP significantly reduced the levels of specific TNF mRNA
(Fig. 6). These results indicate that both neuropeptides exert their
action at a transcriptional level.
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Fig. 6.
VIP and PACAP inhibit TNF
transcription. Raw 264.7 cells (2 × 107 cells)
were stimulated with LPS (1 µg/ml) and incubated with or without VIP
or PACAP (108 M) for 1.5, 3, or 6 h.
Cells incubated with medium alone were used as basal TNF mRNA
level controls. Total RNA was extracted and the expression of TNF
and 18 S mRNA was analyzed by Northern blot analysis. One
representative experiment of three is shown.
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VIP and PACAP Inhibit NF-kB Binding--
Activation and nuclear
translocation of members of the NF-kB/c-Rel family constitutes the
hallmark of macrophage stimulation by proinflammatory cytokines and
bacterial products (37). To investigate whether VIP/PACAP affect NF-kB
nuclear translocation, we used electrophoretic mobility shift assays.
Stimulation of Raw 264.7 cells with LPS led to strong NF-kB binding
compared with unstimulated cells, and treatment with VIP or PACAP
significantly reduced the binding (Fig.
7A). The binding specificity
was confirmed by using homologous (NF-kB) and nonhomologous (CRE)
oligonucleotides as competitors (Fig. 7B). Furthermore,
monospecific anti-p50 and anti-p65 Abs used in supershift experiments
indicated that the LPS-induced kB binding complex was composed
primarily of p50/p65 heterodimers (Fig. 7C).
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Fig. 7.
VIP and PACAP inhibit NF-kB binding.
A, nuclear extracts were prepared from Raw 264.7 cells
(2 × 107 cells) incubated for 2 h with LPS (1 µg/ml) in the presence or absence of VIP or PACAP (108
M). NF-kB binding was assessed by EMSA using a radiolabeled
oligonucleotide containing the murine kB site of the TNF promoter.
B and C, specificity and identification of NF-kB
subunit composition was conducted by the addition of 50-fold excess of
unlabeled homologous (NF-kB) or nonhomologous
(CRE) oligonucleotides (Comp) (B), and
by supershift analysis using polyclonal antibodies to the p50 and p65
subunits of NF-kB (C). The faster migrating band represents
nonspecific protein binding. Similar results were observed in three
independent experiments.
|
|
VIP and PACAP Modulate the Composition of the CRE Binding
Complex--
Recently, it has been established that the CRE site in
the TNF promoter is required for optimal transcription of the TNF gene in monocytes (20). The CRE binding activity is constitutively expressed in unstimulated Raw 264.7 cells, and treatment with LPS in
the presence or absence of VIP/PACAP does not affect the binding (Fig.
8A). The specificity of the
CRE binding activity was confirmed with homologous (CRE) or
nonhomologous (NF-kB) oligonucleotides as competitors (Fig.
8A). Antibody supershift experiments were performed to
determine the composition of the CRE binding complexes. In unstimulated
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.
8B). In contrast, a major supershift by the anti-c-Jun Ab
was evident in cells treated with LPS (Fig. 8B), indicating
the presence of c-Jun in the CRE binding complexes. Treatment of
LPS-stimulated cells with VIP or PACAP led to complexes similar to
those from unstimulated cells, containing CREB and minor amounts, if
any, of c-Jun (Fig. 8B).
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Fig. 8.
VIP and PACAP regulate LPS induction of CRE
binding nuclear factors. A, nuclear extracts were
prepared from Raw 264.7 cells (2 × 107 cells)
incubated for 2 h with LPS (1 µg/ml) in the presence or absence
of VIP or PACAP (108 M). CRE binding activity
was determined by EMSA using a radiolabeled oligonucleotide containing
the murine CRE site from the TNF promoter. Specificity was
determined by the addition of 50-fold excess of unlabeled nonhomologous
(NF-kB) or homologous (CRE) oligonucleotides
(Comp). B, identification of the proteins bound
to the CRE site. Nuclear extracts were preincubated with either
anti-c-Jun or anti-CREB antibodies as described under "Experimental
Procedures" before the addition of the radiolabeled probe. Similar
results were observed in three independent experiments.
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|
VIP and PACAP Inhibit JNK Activity in LPS-stimulated Raw 264.7 Cells--
Phosphorylation of c-Jun at Ser63 and
Ser73 by JNK after LPS stimulation is essential for the
binding of the c-Jun protein to the CRE site (38-40). Because VIP and
PACAP reduce c-Jun in the CRE binding complexes, we investigated
whether VIP inhibits JNK activity. Unstimulated and LPS-stimulated
cells were incubated with or without VIP for 1-6 h, and the presence
of phosphorylated c-Jun was determined by Western blot. LPS-stimulated
cells express high levels of phosphorylated c-Jun in comparison with
unstimulated cells (Fig. 9). VIP reduces
the expression of c-Jun in a time-dependent manner, with
the highest effect at 2 and 3 h after stimulation (Fig. 9).
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Fig. 9.
VIP and PACAP inhibit LPS-stimulated
increases in JNK activity. Raw 264.7 cells (2 × 107 cells) were stimulated with LPS (1 µg/ml) in the
presence or absence of VIP or PACAP (108 M)
for different time periods (1, 2, 3, and 6 h) after which they
were lysed and processed as described under "Experimental
Procedures." Equal amounts of protein were resolved by
SDS-polyacrylamide gel electrophoresis gels and transferred to
nitrocellulose membranes. JNK activity was assayed using a specific
antibody against phosphorylated c-Jun. Cells cultured in medium served
as controls. Phosphorylated c-Jun is indicated by  . MW,
molecular weight markers. Similar results were observed in three
independent experiments.
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Involvement of VIP1R and cAMP in the Effects of VIP on
kB and CRE Binding--
Because the inhibitory effect of VIP on TNF
production is mediated primarily through VIP1R and cAMP, we
determined the effect of the VIP1R antagonist and of the
protein kinase A inhibitor H89 on the changes induced by VIP in kB and
CRE binding complexes. The inhibitory activity of VIP on LPS-mediated
NF-kB binding was completely reversed by the
VIP1R-antagonist (Fig.
10A, lane 4), and
only partially by H89 (Fig. 10A, lane 3).
However, both the VIP1R antagonist and H89 reversed the
changes in the composition of the CRE binding complexes induced by VIP.
In the presence of either VIP1R antagonist or H89 the
supershift patterns returned to the patterns observed for
LPS-stimulated cells in the absence of VIP (Fig. 10B,
lanes 3 and 4 compared with lane 1,
and lanes 8 and 9 compared with lane
6). These results suggest that both the inhibition of NF-kB and
the change in the composition of the CRE binding complexes by VIP are
mediated through the VIP1R, but only the change in the CRE
binding complex is entirely cAMP-dependent. This is
supported by the fact that forskolin (a cAMP inducer) does not affect
NF-kB binding (Fig. 10A, lane 5) but affects CRE binding complexes in the same way as VIP (Fig. 10B,
lane 5 compared with lane 2, and lane
10 compared with lane 7).
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Fig. 10.
Specific receptors and intracellular
pathways involved in the VIP and PACAP regulation of nuclear
factors. A, NF-kB binding. Nuclear extracts prepared
from LPS-stimulated Raw 264.7 cells were incubated with the NF-kB
oligonucleotide and subjected to EMSA. Lane 1: LPS;
lane 2: LPS+VIP; lane 3: LPS+VIP+H89; lane
4: LPS+VIP+VIP1R antagonist; lane 5:
LPS+FK. B, CRE binding complex. Nuclear extracts prepared
from LPS-stimulated Raw 264.7 cells were incubated with the CRE
oligonucleotide and subjected to EMSA. In all three panels, lane
1: LPS; lane 2: LPS+VIP; lane 3:
LPS+VIP+VIP1R antagonist; lane 4: LPS+VIP+H89;
lane 5: LPS+FK. First panel, supershift with
anti-c-Jun Ab; second panel, supershift with anti-CREB Ab;
third panel, no Ab.
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The cAMP-independent Transduction Pathway Is Involved in the
Inhibition of NF-kB Binding--
Because both a
cAMP-dependent and a cAMP-independent pathway appear to be
involved in the inhibition of TNF production by VIP, we investigated
the possible relationship between the two pathways and the regulation
of the kB and CRE binding complexes. Because SNV inhibits TNF
production without the involvement of cAMP, we determined its effect on
the kB and CRE binding nuclear factors. Similar to VIP, SNV inhibits
NF-kB binding in LPS-stimulated cells, although to a lesser degree
(Fig. 11A). In contrast, SNV does not change the composition of the CRE binding complexes in LPS-stimulated cells (Fig. 11B). This suggests that the
cAMP-independent pathway is involved solely in the inhibition of NF-kB
binding. This conclusion is supported by the effect of SANV, the
lipophilic VIP antagonist that does not affect cAMP induction (22).
SANV reverses the inhibitory activity of VIP on NF-kB binding, without affecting the regulatory effect of VIP on the composition of the CRE
binding complexes (Fig. 11, A and B).
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Fig. 11.
Different transduction pathways are involved
in the inhibition of NF-kB binding and in the regulation of the
composition of CRE binding complexes. A, NF-kB
binding. Nuclear extracts prepared from LPS-stimulated Raw 264.7 cells
were incubated with the NF-kB oligonucleotide and subjected to EMSA.
Lane 1, LPS; lane 2, LPS+VIP (108
M); lane 3, LPS+SNV (107
M); lane 4, LPS+VIP (108
M) +SANV (106 M). B,
CRE binding complex. Nuclear extracts prepared from LPS-stimulated Raw
264.7 cells were incubated with the CRE oligonucleotide and subjected
to EMSA. In all three panels, lane 1, LPS; lane
2, LPS+VIP (108 M); lane 3, LPS+SNV 107 M); lane 4, LPS+VIP
(108 M)+SANV (106
M). First panel, supershift with anti-c-Jun Ab;
second panel, supershift with anti-CREB Ab; third
panel, no Ab. One representative experiment of four is
shown.
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|
| |
DISCUSSION |
VIP and PACAP are two multifunctional neuropeptides with
regulatory roles in inflammation (reviewed in Ref. 10). We have recently described that VIP and PACAP protect mice from high
endotoxemia and inhibit in vitro and in vivo IL-6
and TNF production by murine peritoneal macrophages
(11).2,3 Here we extend these
studies to the molecular mechanisms involved in the inhibitory effect
of VIP/PACAP on TNF production. Our results indicate that VIP/PACAP
inhibit LPS-induced TNF production in Raw 264.7 murine macrophages.
The inhibitory effect is dose-dependent within a wide range
of neuropeptide concentrations (107-1011
M), with the maximum effect being observed at
108 M. This is the dose range at which VIP
and PACAP modulate several immunological functions (10, 41).
Similar to the effect on other cytokines such as IL-2, IL-6, and IL-10
(11, 42, 43), the inhibition of TNF 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 (44, 45). Peritoneal macrophages were previously
shown to express VIP1R and PACAP-R mRNA, and both high
and low affinity VIP/PACAP binding sites (16, 17). Here we report that
LPS-stimulated Raw 264.7 macrophages express mRNA for all three
VIP/PACAP receptors, although the membrane expression of the three
receptors remains to be demonstrated. Our agonist studies suggest that
VIP1R is the major mediator of the VIP/PACAP inhibitory
effect on TNF (60% inhibition observed with the VIP1R
agonist in comparison with 20-25% for VIP2R and PACAP-R
agonists). This is in agreement with Dewit et al. (13) who
reported a maximal 34% inhibition of TNF by 105
M Ro 25-1553 (a VIP2R agonist) in human blood
monocytes. If the VIP2R is expressed only in activated
macrophages, as the reverse transcription-PCR results suggest, the lack
of effectiveness for the Ro compounds may be because of a lack of
appropriate receptors during the early culture period. The role of
VIP1R as the major player in mediating the effect of
VIP/PACAP on TNF production is also supported by the fact that a
VIP1R antagonist, but not PACAP6-38, an antagonist specific
for both PACAP-R and VIP2R (33), reverses the inhibitory
effect of VIP/PACAP. Also, the VIP1R antagonist blocked the
effect of VIP/PACAP on both NF-kB and c-Jun/CREB binding to the TNF
promoter, supporting the involvement of the VIP1R in the
regulatory effect of VIP/PACAP on TNF gene expression.
The VIP1R is coupled primarily to the adenylate cyclase
system (14). TNF production is inhibited by agents that increase intracellular cAMP levels, and stimulated by the activation of the
protein kinase C pathway (2, 4, 46-48). In the present study, H89, a
potent and selective inhibitor of protein kinase A reversed the
inhibitory effect of VIP/PACAP on TNF production, suggesting that
VIP/PACAP inhibit TNF production in Raw 264.7 cells through protein
kinase A activation and elevation of cAMP levels. However, because the
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 IL-2 and IL-10
production.4 The existence of
a second cAMP-independent pathway is supported by the effect of a
lipophilic VIP agonist, SNV, which does not induce cAMP (22). SNV
inhibits TNF production in Raw 264.7 cells, and, as expected in the
absence of cAMP induction, the inhibitory effect is not affected by
H89. The fact that SNV does not contribute to the inhibitory activity
of VIP/PACAP, although it adds to the effect of other cAMP-inducing
agents such as PGE2 or forskolin, suggests that indeed a
second cAMP-independent pathway may function in the transduction of the
VIP/PACAP signal. The nature of this second transduction pathway
remains to be determined.
Previous experiments regarding VIP modulation of cytokine expression,
indicated different molecular mechanisms, i.e.
transcriptional regulation for IL-2, IL-6, and IL-10 versus
post-transcriptional regulation for IL-4 (11, 43, 49). The present
study indicates that the inhibitory effect of VIP and PACAP on TNF
production occurs at a transcriptional level. The regulation of the
TNF gene transcription is complex, and involves multiple
cis-acting elements. Transcriptional regulation by LPS of
the TNF gene has been shown to involve kB sequence motifs and
transcriptional factors from the Rel family (18-21, 37, 50, 51). In
mammalian cells, the Rel family includes NF-kB1 (p50), RelA (p65),
c-Rel, RelB, and NF-kB2 (p50B, p52) (37). NF-kB consists mostly of
p50/p65 heterodimers, which are complexed to the inhibitor IkB in the cytoplasm of unstimulated cells; stimuli such as LPS and
proinflammatory cytokines induce the phosphorylation of IkB, followed
by the release and subsequent nuclear translocation of the p50/p65
heterodimers, which bind to regulatory sequences in a variety of target
genes (37). The studies presented here indicate that VIP and PACAP inhibit NF-kB binding in LPS-stimulated Raw 264.7 cells. The NF-kB complex induced by LPS in macrophages was partly supershifted by either
anti-p50 or anti-p65 Ab and fully shifted by a combination of these two
Abs, suggesting that the NF-kB complex responsible for TNF induction
by LPS consisted of both p50 and p65 subunits. It remains to be
determined whether VIP/PACAP-mediated NF-kB nuclear translocation
inhibition results from an increase in IkB protein levels, a decrease
in IkB degradation, and/or inhibition in IkB phosphorylation, as in the
case of other anti-inflammatory agents, such as IL-11, IL-10,
transforming growth factor-1, glucocorticoids, and antioxidants (6,
52-56).
In addition to the kB sites, a CRE site was recently identified as
necessary for maximal LPS induction of the TNF gene in human
monocytes (20). Similar to human monocytes, CRE binding activity is
constitutively expressed in Raw 246.7 cells. LPS and VIP/PACAP
treatment does not increase or decrease the amount of CRE binding;
however, supershift experiments with anti-c-Jun Abs indicate that LPS
induces a marked increase in c-Jun binding, similar again to human
monocytes (20). In contrast, VIP/PACAP reduce c-Jun binding to levels
present in the unstimulated cells. This suggests that the inhibitory
effect of VIP/PACAP on TNF gene expression is mediated, at least
partially, through a change in the composition of the CRE binding
complexes. Because c-Jun phosphorylation by JNK results in both an
amplification of c-Jun synthesis and an increase in transactivating
activity (40, 57), the effect of VIP/PACAP on c-Jun may be mediated
through an effect on JNK. This is indeed the case, because VIP inhibits
JNK activity in LPS-stimulated Raw 264.7 cells. The effect of VIP on
JNK is in agreement with the previously reported selective inhibition of JNK in T lymphocytes by cAMP-elevating agents (58). In terms of the
molecular mechanisms involved in the inhibition of TNF gene
expression, VIP resembles glucocorticoids more than cytokines. IL-10
and IL-11, which inhibit TNF expression, appear to act solely on
NF-kB (6, 52). In contrast, dexamethasone down-regulates both NF-kB and
AP-1 binding (59), and this possible synergistic effect may explain why
glucocorticoids are more potent TNF inhibitors.
We investigated the relationship between the cAMP-dependent
and cAMP-independent transduction pathways for the inhibitory effect of
VIP/PACAP on TNF expression and the effect on NF-kB and CRE binding
activities. Both VIP and forskolin induced similar changes in the CRE
binding complexes, and H89, a specific protein kinase A inhibitor,
reversed the effect of VIP on the composition of the CRE binding
complex. This suggests that VIP/PACAP-elicited cAMP controls changes in
the composition of the CRE binding complexes, most probably by
increasing protein kinase A-dependent CREB phosphorylation and decreasing JNK-dependent c-Jun phosphorylation. In
contrast, increases in cAMP do not appear to directly affect NF-kB
binding. Forskolin, a strict cAMP inducer, does not affect NF-kB
binding, and H89 reverses only partially the inhibitory effect of VIP. Also SNV, a VIP agonist that does not induce cAMP, inhibits NF-kB binding without affecting the composition of the CRE binding complex. The fact that cAMP-inducing agents do not affect NF-kB binding, although they inhibit kB transcriptional activation, has been previously reported (60). The inhibition of the NF-kB transcriptional activity could result from higher amounts of CRE-bound CREB competing with NF-kB for limiting amounts of the coactivator CBP (61). Recently
VIP and PACAP were reported to increase CREB phosphorylation and
CREB-regulated transcription in several cell types (62-64). Therefore,
an additional mechanism in the VIP/PACAP inhibition of kB-mediated
transactivation of the TNF gene may involve the competition between
NF-kB and CREB for CBP.
In conclusion, we have shown that the binding of VIP and PACAP to
VIP1R inhibits TNF production at a transcriptional level in LPS-stimulated Raw 264.7 macrophages through two intracellular pathways, a cAMP-dependent pathway that preferentially
increases CREB versus c-Jun binding to the CRE site, and a
cAMP-independent pathway that inhibits the binding of NF-kB (Fig.
12). The inhibition of TNF
transcription by VIP/PACAP may have significant therapeutic potential,
because this proinflammatory cytokine plays a central role in endotoxic
shock, multiple sclerosis, cerebral malaria, and various inflammatory
diseases (2, 4). Also, the effect of VIP/PACAP on NF-kB binding may be
of therapeutic significance, because NF-kB has been proposed as a
target for the treatment of inflammatory conditions such as rheumatoid
arthritis and inflammatory bowel disease (37).
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Fig. 12.
Model for the inhibitory effect of VIP/PACAP
on TNF gene expression. Binding of VIP to
VIP1R initiates two transduction pathways. The
cAMP-dependent pathway, mimicked by FK, leads to JNK
inhibition and an increase in CREB phosphorylation, resulting in the
alteration of the CRE binding complexes from high Jun/low CREB in
LPS-stimulated cells to low Jun/high CREB in LPS+VIP-treated cells. The
cAMP-independent pathway, mimicked by SNV, leads to an inhibition of
NF-kB nuclear translocation, resulting in reduced NF-kB binding. In
addition, higher amounts of VIP-induced CREB compete with NF-kB for
limited amounts of the coactivator CBP. The decrease in c-Jun and
NF-kB, and the sequestering of CBP leads to the inhibition of TNF
transcription.
|
|
| |
ACKNOWLEDGEMENTS |
We thank Dr. David Pozo (University of
Sevilla, Sevilla, Spain) for the TNF oligonucleotide probe, Dr.
Patrick Robberecht (Universite Libre de Bruxelles, Brussels, Belgium)
for the VIP1R agonist and antagonist, Drs. David Bolin and
Ann Welton (Hoffman-LaRoche, Inc.) for the VIP2R agonist Ro
25-1553, and Dr. Ethan Lerner (Massachusetts General Hospital,
Charlestown, MA) for the PACAP-R agonist maxadilan.
| |
FOOTNOTES |
*
This work was supported by Grants PHS AI 41786-01 and Busch
Biomedical Award 96-98 (to D. G.), Grant PB94-0310 (to R. P. G.), and postdoctoral fellowship from the Spanish Ministry of
Education and Science (to M. D.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Rutgers University,
Dept. of Biological Sciences, 101 Warren St., Newark, NJ 07102. Tel.:
973-353-1162; Fax: 973-353-1007; E-mail:
dganea{at}andromeda.rutgers.edu.
The abbreviations used are:
LPS, lipopolysaccharide; TNF, tumor necrosis factor ; IL, interleukin; VIP, vasoactive intestinal peptide; PACAP, pituitary adenylate
cyclase-activating polypeptide; CRE, cAMP responsive element; CREB, CRE-binding protein; CBP, CREB-binding protein; PGE2, prostaglandin E2; NF-kB, nuclear factor kB; ELISA, enzyme-linked immunosorbent assay; Ab, antibody; PCR, polymerase chain
reaction; bp, base pair(s); H89, N-[2-(p-bromocinnamyl-amino)ethyl]-5-iso-quinolinesulfonamide; EMSA, electrophoretic mobility shift assay; FK, forskolin.
2
Delgado, M., Martinez, C., Pozo, D., Leceta, J.,
Calvo, J. R., Ganea, D., and Gomariz, R. P. (1998) J. Immunol., in press.
3
M. Delgado, D. Pozo, C. Martinez, J. Leceta,
J. R. Calvo, D. Ganea, and R. P. Gomariz, manuscript
submitted for publication.
4
H.-Y. Wang, X. Jiang, I. Gozes, M. Fridkin,
D. E. Brenneman, and D. Ganea, manuscript submitted for publication.
| |
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Top
Abstract
Introduction
