Vasoactive intestinal peptide (VIP), a neuropeptide present in the lymphoid microenvironment, and the structurally related pituitary adenylate cyclase-activating polypeptide (PACAP) act as potent anti-inflammatory agents that inhibit the function of activated macrophages and TH cells. Previous reports showed that VIP/PACAP inhibit IL-6 and TNF-α production in LPS-stimulated macrophages. The present study reports on the effect of VIP/PACAP on IL-10 production. Although VIP/PACAP do not induce IL-10 by themselves, they enhance IL-10 production in LPS-stimulated macrophages. The specific VPAC1 receptor mediates the stimulatory effect of VIP/PACAP, and cAMP is the major second messenger involved. VIP/PACAP increase IL-10 mRNA in LPS-stimulated cells, and the effect of transcriptional and protein synthesis inhibitors indicates de novo IL-10 production. Electromobility shift assays show that VIP/PACAP induce an increase in nuclear cAMP response element (CRE)-binding complexes, with CRE binding protein as the major active component. Treatments with either a VPAC1 antagonist or a protein kinase A inhibitor abolish IL-10 stimulation and, concomitantly, the increase in CRE binding. Effects similar to the in vitro stimulation of IL-10 were obtained in vivo in mice treated with LPS and VIP or PACAP. The neuropeptides induce increased levels of IL-10 in both serum and peritoneal fluid, and increased expression of the IL-10 mRNA in peritoneal exudate cells. The stimulation of IL-10 production in activated macrophages represents a novel anti-inflammatory activity of VIP and PACAP, which presumably acts in vivo in conjunction with the inhibition of proinflammatory cytokines such as IL-6 and TNF-α to reduce the magnitude of the immune response.

The generation of an immune response involves the activation of effector cells such as macrophages, neutrophils, and T lymphocytes and the subsequent production of cytokines, chemokines, and reactive oxygen and nitrogen intermediates. The activated macrophages are widely recognized as cells that play an important role in inflammatory processes as well as in the initiation, maintenance, and control of specific immune responses. In response to LPS and other activating agents, macrophages secrete nitric oxide and proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-12, and immunomodulatory cytokines such as TGF-β1 and IL-10 (1). Since the intensity and duration of an inflammatory process depend on the local balance between pro- and anti-inflammatory factors, the so-called macrophage-deactivating factors have received considerable attention lately (2, 3, 4, 5, 6).

Vasoactive intestinal peptide (VIP)3 and the pituitary adenylate cyclase-activating polypeptide (PACAP) are two multifunctional neuropeptides whose primary immunomodulatory function is anti-inflammatory in nature. VIP and PACAP have been shown to inhibit cytokine production and proliferation in T cells (reviewed in 7 and to inhibit macrophage phagocytosis, respiratory burst, and chemotaxis (reviewed in 8 . In agreement with their anti-inflammatory role, VIP/PACAP were recently reported to inhibit IL-6 and TNF-α production in LPS-stimulated macrophages (9, 10, 11, 63), and to protect mice from endotoxic shock, presumably through the inhibition of endogenous TNF-α and IL-6 (12).

The immunological actions of VIP and PACAP are exerted through a family of VIP/PACAP receptors that have been recently reclassified (13): VPAC1 and VPAC2, which exhibit similar affinities for the two neuropeptides and activate primarily the adenylate cyclase system, and PAC1, which exhibits a 300- to 1000-fold higher affinity for PACAP than for VIP and activates both the adenylate cyclase and phospholipase C systems (reviewed in 14 . Murine peritoneal macrophages were shown to possess VPAC1 and PAC1 (15, 16, 17).

IL-10, one of the major anti-inflammatory cytokines, was initially described as a Th2 product that inhibits the secretion of Th1-derived cytokines through the down-regulation of the Ag-presenting function of professional APCs (18, 19). In addition to T cells, activated monocytes/macrophages serve as a major IL-10 source, especially in response to LPS stimulation (20, 21). IL-10 inhibits several macrophage functions, such as oxidative burst, phagocytosis, nitric oxide production, and cytokine production (4, 20, 22, 23, 24, 25).

Based on their effect on macrophage function, both the neuropeptides VIP/PACAP and the cytokine IL-10 could be classified as macrophage-deactivating factors. However, in addition to their direct inhibitory effect on cytokine production by activated T cells and macrophages, VIP/PACAP may contribute to the down-regulation of the immune response through the induction of anti-inflammatory cytokines, particularly IL-10. In the present study we examine the in vitro and in vivo effects of VIP/PACAP on IL-10 protein and mRNA levels in LPS-stimulated peritoneal macrophages and investigate the molecular mechanisms involved.

Synthetic VIP, PACAP38, VIP1–12, and VIP10–28 were purchased from Novabiochem (Laufelfingen, Switzerland). The VPAC1 antagonist [Ac-His1,d-Phe2,K15,R16,L27]VIP3–7-GRF8–27 and the VPAC1 agonist [K15,R16,L27]VIP1–7-GRF8–27 were donated by Dr. Patrick Robberecht (Universite Libre de Bruxelles, Brussels, Belgium). The VPAC2 agonist Ro 25–1553 Ac-[Glu8,Lys12,Nle17,Ala19,Asp25,Leu26,Lys27,28,Gly29,30,Thr31]VIP- cyclo21–25 was a gift from Drs. Ann Welton and David R. Bolin (Hoffmann-La Roche, Nutley, NJ). The synthetic PAC1 agonist maxadilan was a gift from Dr. Ethan A. Lerner (Massachusetts General Hospital, Charlestown, MA). The VPAC2/PAC1 antagonist PACAP6–38, secretin, and glucagon were obtained from Peninsula Laboratories (Belmont, CA). Oligonucleotides were synthesized by the Oligonucleotide Synthesis Service of Rutgers University (Newark, NJ). Murine rIL-10 and capture and biotinylated anti-murine IL-10 mAbs were purchased from PharMingen (San Diego, CA). LPS (from Escherichia coli 055:B5), calphostin C, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS substrate), forskolin, PGE2, protease inhibitors, PMSF, EDTA, glycine, protein G-agarose, glycerol, EGTA, DTT, and avidin-peroxidase were purchased from Sigma (St. Louis, MO), and N-[2-(p-bromocinnamyl-amino)ethyl]-5-iso-quinolinesulfonamide (H89) was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Abs against CREB and c-Jun were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All reagents were dissolved in endotoxin-free distilled water and diluted in serum-free RPMI 1640 (certified as endotoxin-free; Life Technologies, Gaithersburg, MD).

Mouse peritoneal macrophages were elicited by i.p. injection of 2 ml of 4% Brewer’s thioglycolate medium (Difco, Detroit, MI) into male BALB/c mice (aged 6–10 wk; National Cancer Institute, Frederick, MD). Peritoneal exudate cells were obtained 72 h after injection by peritoneal lavage with 4 ml of ice-cold RPMI 1640 medium. Peritoneal exudate cells containing lymphocytes and macrophages were washed twice and resuspended in ice-cold RPMI 1640 medium supplemented with 2% heat-inactivated FCS (Atlanta Biologicals, Norcross, GA; certified as containing endotoxin levels <10 EU/ml) containing 10 mM HEPES buffer, 1 mM pyruvate, 0.1 M nonessential amino acids, 2 mM glutamine, 50 mM 2-ME, 100 U/ml penicillin, and 10 μg/ml streptomycin (RPMI 1640 complete medium). Cells were seeded in flat-bottom 96-well microtiter plates (Corning Glass, Corning, NY) at 8 × 104 cells/well in a final volume of 200 μl. The cells were incubated at 37°C for 2 h to allow adherence to plastic, and nonadherent cells were removed by repeated washing with RPMI 1640 medium. At least 96% of the adherent cells were macrophages as judged by morphologic and phagocytic criteria. Macrophage monolayers were incubated with RPMI complete medium and stimulated with LPS (0.5 μg/ml) in the presence or the absence of VIP or PACAP38 (from 10−12 to 10−6 M) at 37°C in a humidified incubator with 5% CO2. Cell-free supernatants were harvested at the designated time points and kept frozen (−20°C) until determination of IL-10 production by ELISA.

Male BALB/c mice (6–10 wk old) received a single i.p. dose of LPS (100 μg/mouse) in the presence or the absence of different amounts of VIP or PACAP (from 0.5–10 nmol/mouse). After different time periods (2–8 h), blood was removed through cardiac puncture, and peritoneal exudate was obtained by peritoneal lavage with 4 ml of ice-cold RPMI 1640 medium. The blood samples were allowed to clot for 1 h at room temperature; serum was obtained and kept frozen until used in IL-10 ELISA. The peritoneal suspension was centrifuged 5 min at 1800 × g, and cell-free supernatants (peritoneal fluid) were harvested and assayed for IL-10 production. The peritoneal cells were subjected to Northern blot analyses as described below.

The amounts of IL-10 present in culture supernatants, peritoneal fluid, and serum were determined using the murine IL-10 sandwich ELISA essentially as described previously (26), with the IL-10-specific mAb JES3–9D7 as capture Ab and the biotinylated anti-IL-10 mAb JES-12G8 as detection Ab. The sensitivity of the IL-10 ELISA was 10 pg/ml. The assay was specific for IL-10, with other recombinant cytokines (IL-1, IL-2, IL-4, IL-7, IL-6, IL-12, and IFN-γ) not binding above background levels.

Northern blot analysis was performed according to standard methods. Macrophage monolayers (107 cells/well in six-well tissue culture plates) were cultured in 5 ml of medium and stimulated with LPS (0.5 μg/ml) in the absence or the presence of VIP and PACAP (10−8 M) for different time periods at 37°C. Total RNA was extracted by the acid guanidinium-phenol-chloroform method, electrophoresed on 1.2% agarose-formaldehyde gels, transferred to Nytran membranes (Schleicher and Schuell, Keene, NJ), and cross-linked to the nylon membrane using UV light. The probes for murine IL-10 and β-actin were generated by RT-PCR as described previously (26). Oligonucleotides were end labeled with [γ-32P]ATP using T4 polynucleotide kinase. The RNA-containing membranes were prehybridized for 16 h at 42°C and then hybridized at 60°C for 16 h with the appropriate probes. The membranes were washed twice in 2× SSC containing 0.1% SDS at room temperature (20 min each time), once at 37°C for 20 min, and once in 0.1× SSC containing 0.1% SDS at 50°C (20 min). The prehybridization and hybridization buffers were purchased from 5 Prime-3 Prime (Boulder, CO). The membranes were exposed to x-ray films (Eastman Kodak, Rochester, NY). Signal quantitation was performed in a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA).

Nuclear extracts were prepared by a mini-extraction procedure. Briefly, peritoneal macrophages were plated at a density of 107 cells in six-well plates, stimulated, washed twice with ice-cold PBS/0.1% BSA, and scraped off the dishes. The cell pellets were homogenized with 0.4 ml of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 1 mM NaN3). After 15 min on ice, Nonidet P-40 was added to a 0.5% final concentration, the tubes were gently vortexed for 15 s, and nuclei were sedimented and separated from cytosol by centrifugation at 12,000 × g for 40 s. The pelleted nuclei were washed once with 0.2 ml of ice-cold buffer A, and the soluble nuclear proteins were released with 0.1 ml of buffer C (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 1 mM NaN3). After incubation for 30 min on ice, the nuclear proteins were separated from the remaining nuclei by centrifugation for 10 min at 14,000 rpm at 4°C. The protein concentration in the supernatants was determined by the Bradford method, and aliquots were stored at −80°C for later use in EMSAs.

Oligonucleotides corresponding to the CRE (5′-TTTATCCACGTCATTATGACC-3′; nucleotides 1201–1221), and NF-κB (5′-CTCTCGGGGTTTCCTTTGGG-3′; nucleotides 1069–1088) motifs of the IL-10 promoter were synthesized (27). Oligonucleotides were annealed by incubation for 5 min at 85°C in 10 mM Tris-HCl (pH 8.0), 5 mM NaCl, 10 mM MgCl2, and 1 mM DTT. Fifty nanograms of the double-stranded oligonucleotides were end labeled with [γ-32P]ATP using T4 polynucleotide kinase. For EMSAs with macrophage nuclear extracts, 20,000–50,000 cpm of the double-stranded oligonucleotide, corresponding to approximately 0.5 ng, was used for each reaction. Binding reaction mixtures (15 μl) were set up containing 0.5–1 ng of DNA probe, 5 μg of nuclear extract, 2 μg of poly(dI-dC)·poly(dI-dC), and binding buffer (50 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 5% glycerol, and 10 mM Tris-HCl, pH 7.5). The reaction was incubated on ice for 15 min before adding the probe, followed by 20 min at room temperature. Samples were loaded onto 4% nondenaturing polyacrylamide gel and electrophoresed in TGE buffer (50 mM Tris-HCl (pH 7.5), 0.38 M glycine, and 2 mM EDTA) at 100 V, followed by transfer to Whatman paper (Clifton, NJ), drying under vacuum at 80°C, and autoradiography. In competition and Ab supershift experiments, the nuclear extracts were incubated for 15 min at room temperature with Ab (1 μg) or competing oligonucleotide (50-fold excess) before addition of the labeled probe.

All values are expressed as the mean ± SD of the number of experiments. In each experiment samples were assayed in duplicate. Comparisons between groups were made using Student’s t test followed by Scheffe’s F test, with p < 0.05 as the minimum significant level.

Peritoneal macrophages cultured in the presence of VIP or PACAP (from 10−7 to 10−11 M) did not secrete detectable levels of IL-10. However, the addition of VIP/PACAP to LPS-stimulated cultures resulted in a significant increase in IL-10 production. VIP and PACAP dose-dependently increased the amount of IL-10 released in response to LPS, showing maximal effects at 10−6–10−8 M (Fig. 1,A). The time curves indicate that VIP and PACAP stimulate IL-10 release as early as 4 h, with a maximum stimulatory effect at 24 h (Fig. 1 B).

FIGURE 1.

VIP and PACAP stimulate IL-10 production by LPS-treated macrophages. A, Dose-response curve for the stimulatory effect of VIP and PACAP on IL-10 production. Peritoneal macrophages were stimulated with LPS (0.5 μg/ml) and a concentration range of either VIP or PACAP for 24 h. The IL-10 contents of culture supernatants were determined by ELISA. The dotted line represents the IL-10 value in LPS-stimulated macrophages cultured in the absence of VIP/PACAP. B, Time course for the stimulation of IL-10 production by VIP and PACAP. Macrophages were stimulated with LPS (0.5 μg/ml) in the absence or the presence of 10−8 M VIP or PACAP, and supernatants harvested at different times were assayed for IL-10 by ELISA. Control cultures were incubated with LPS alone. For A and B, cells cultured in the absence of LPS with or without VIP/PACAP did not produce detectable levels of IL-10 (<10 pg/ml). Each result is the mean ± SD of four separate experiments. Each sample was assayed in duplicate. ∗, p < 0.001 with respect to control cultures with LPS alone.

FIGURE 1.

VIP and PACAP stimulate IL-10 production by LPS-treated macrophages. A, Dose-response curve for the stimulatory effect of VIP and PACAP on IL-10 production. Peritoneal macrophages were stimulated with LPS (0.5 μg/ml) and a concentration range of either VIP or PACAP for 24 h. The IL-10 contents of culture supernatants were determined by ELISA. The dotted line represents the IL-10 value in LPS-stimulated macrophages cultured in the absence of VIP/PACAP. B, Time course for the stimulation of IL-10 production by VIP and PACAP. Macrophages were stimulated with LPS (0.5 μg/ml) in the absence or the presence of 10−8 M VIP or PACAP, and supernatants harvested at different times were assayed for IL-10 by ELISA. Control cultures were incubated with LPS alone. For A and B, cells cultured in the absence of LPS with or without VIP/PACAP did not produce detectable levels of IL-10 (<10 pg/ml). Each result is the mean ± SD of four separate experiments. Each sample was assayed in duplicate. ∗, p < 0.001 with respect to control cultures with LPS alone.

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The experiments reported above were performed with peritoneal adherent cells. Since peritoneal lymphocytes, especially CD5+ B cells, which secrete IL-10 (28), may contaminate the adherent cell population, we determined the effect of VIP/PACAP on the IL-10 production by LPS-stimulated nonadherent peritoneal cells. Nonadherent peritoneal cells obtained after the 2-h incubation in tissue culture plates were stimulated with LPS in the presence or the absence of VIP and PACAP. Supernatants collected 24 h later showed no detectable IL-10 (data from three experiments, not shown). In addition, we found no differences in the amount of IL-10 produced by thioglycolate-induced, freshly isolated peritoneal cell suspensions (∼60–80% macrophages, 20–40% lymphocytes) and monolayer cell preparations containing about 96% macrophages, confirming that the peritoneal lymphocytes do not contribute to the IL-10 production in response to LPS.

Next we investigated whether the stimulatory effect of VIP/PACAP could be related to the occupancy of specific receptors. First, we compared the effect of VIP/PACAP to that of secretin, glucagon, and the VIP and PACAP fragments VIP1–12, VIP10–28, and PACAP6–38. IL-10 production was not affected by different concentrations of secretin and glucagon (10−7–10−9 M; Fig. 2,A). The two VIP fragments and PACAP6–38 failed to stimulate IL-10 production, suggesting that intact VIP and PACAP molecules are required for their stimulatory activity (Fig. 2 A).

FIGURE 2.

The effect of VIP/PACAP is mediated through VPAC1. A and B, Comparative effects of VIP, PACAP38, VIP-related peptides, VIP and PACAP fragments, and VIP and PACAP agonists on IL-10 production. Peritoneal macrophages were stimulated with LPS (0.5 μg/ml) in the presence or the absence of different concentrations of secretin, glucagon, VIP1–12, VIP10–28, and PACAP6–38 (A) or maxadilan (a PAC1 agonist), Ro 25–1553 (a VPAC2 agonist), and [K15,R16,L27]VIP1–7-GRF8–27 (a VPAC1 agonist; B). Supernatants were collected 24 h later and assayed for IL-10 by ELISA. Each result is the mean ± SD of five experiments. Each sample was assayed in duplicate. ∗, p < 0.001 with respect to control cultures with LPS alone. C and D, Effects of PAC1 and VPAC antagonists on the stimulatory effect of VIP and PACAP. Peritoneal macrophages were stimulated with LPS (0.5 μg/ml) and treated simultaneously with VIP or PACAP (10−8 M) and different concentrations of the VPAC1 antagonist [Ac-His1,d-Phe2,K15,R16, L27]VIP3–7-GRF8–27 (C) or the PAC1/VPAC2 antagonist (PACAP6–38; D). Supernatants were collected 24 h later and assayed for IL-10 by ELISA. The VPAC1 antagonist (10−6 M) and PACAP6–38 (10−6 M) did not affect IL-10 production in LPS-treated macrophages (3166 ± 243 pg/ml for VPAC1 and 3055 ± 115 pg/ml for PACAP6–38). The dotted line in C represents control values from cultures incubated with LPS alone (3033 ± 127 pg/ml). Each result is the mean ± SD of four experiments. Each sample was assayed in duplicate. ∗, p < 0.001 compared with samples treated with neuropeptides and without antagonists. E, Effect of the VPAC1 agonist on the stimulatory activity of VIP/PACAP. Macrophages were stimulated with LPS (0.5 μg/ml) and were treated with the VPAC1 agonist (100 nM) in the presence or the absence of VIP or PACAP (10−8 M). Supernatants were collected 24 h later and assayed for IL-10. The percent stimulation was calculated by comparison with controls containing LPS alone. Results are the mean ± SD of four experiments.

FIGURE 2.

The effect of VIP/PACAP is mediated through VPAC1. A and B, Comparative effects of VIP, PACAP38, VIP-related peptides, VIP and PACAP fragments, and VIP and PACAP agonists on IL-10 production. Peritoneal macrophages were stimulated with LPS (0.5 μg/ml) in the presence or the absence of different concentrations of secretin, glucagon, VIP1–12, VIP10–28, and PACAP6–38 (A) or maxadilan (a PAC1 agonist), Ro 25–1553 (a VPAC2 agonist), and [K15,R16,L27]VIP1–7-GRF8–27 (a VPAC1 agonist; B). Supernatants were collected 24 h later and assayed for IL-10 by ELISA. Each result is the mean ± SD of five experiments. Each sample was assayed in duplicate. ∗, p < 0.001 with respect to control cultures with LPS alone. C and D, Effects of PAC1 and VPAC antagonists on the stimulatory effect of VIP and PACAP. Peritoneal macrophages were stimulated with LPS (0.5 μg/ml) and treated simultaneously with VIP or PACAP (10−8 M) and different concentrations of the VPAC1 antagonist [Ac-His1,d-Phe2,K15,R16, L27]VIP3–7-GRF8–27 (C) or the PAC1/VPAC2 antagonist (PACAP6–38; D). Supernatants were collected 24 h later and assayed for IL-10 by ELISA. The VPAC1 antagonist (10−6 M) and PACAP6–38 (10−6 M) did not affect IL-10 production in LPS-treated macrophages (3166 ± 243 pg/ml for VPAC1 and 3055 ± 115 pg/ml for PACAP6–38). The dotted line in C represents control values from cultures incubated with LPS alone (3033 ± 127 pg/ml). Each result is the mean ± SD of four experiments. Each sample was assayed in duplicate. ∗, p < 0.001 compared with samples treated with neuropeptides and without antagonists. E, Effect of the VPAC1 agonist on the stimulatory activity of VIP/PACAP. Macrophages were stimulated with LPS (0.5 μg/ml) and were treated with the VPAC1 agonist (100 nM) in the presence or the absence of VIP or PACAP (10−8 M). Supernatants were collected 24 h later and assayed for IL-10. The percent stimulation was calculated by comparison with controls containing LPS alone. Results are the mean ± SD of four experiments.

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The nature of the VIP/PACAP receptors involved was investigated using specific receptor agonists and antagonists. We determined the effect of a newly described VPAC1 agonist (29), a VPAC2 agonist (Ro 25–1553) (30), and maxadilan, a specific PAC1 agonist (31), on LPS-induced IL-10 production. The VPAC1 agonist, but not the VPAC2 and PAC1 agonists, stimulated IL-10 release with a potency similar to that of VIP/PACAP (300% stimulation; Fig. 2,B). In addition, we investigated the ability of PACAP6–38, an antagonist specific for PAC1 and to a lesser degree for VPAC2 (32), and of a specific VPAC1 antagonist (33) to reverse the effects of VIP and PACAP. The VPAC1 antagonist reversed the effect of VIP/PACAP in a dose-dependent manner (Fig. 2,C). In contrast, PACAP6–38 did not reverse the stimulatory effect of VIP and PACAP (Fig. 2,D). Neither the VPAC1 antagonist nor PACAP6–38 significantly affected IL-10 levels in LPS-stimulated cells (Fig. 2, C and D). The simultaneous addition of VIP/PACAP and the VPAC1 agonist did not result in an additive effect (Fig. 2 E). Together these results confirm the specificity of the VIP and PACAP stimulatory effect and suggest that both neuropeptides exert their action through VPAC1.

In the experiments described to date VIP and PACAP were added at the same time as LPS. To determine the time interval required for the stimulatory effect of VIP/PACAP, we stimulated peritoneal macrophages with LPS and added 10−8 M VIP or PACAP at later times (from 0–18 h). Supernatants were collected 24 h after the addition of VIP/PACAP and were assayed for IL-10 production. The IL-10 levels were compared with controls containing LPS and no VIP/PACAP, which were harvested at similar time points as the VIP/PACAP-containing cultures. The addition of VIP and PACAP up to 12 h after LPS stimulation resulted in significant levels of stimulation (280–355%; Fig. 3,A). Later additions (18 h) resulted in a lesser degree of stimulation (Fig. 3,A). To determine the minimum time required for the stimulatory effect of VIP and PACAP, peritoneal macrophages were cultured with LPS in the presence or the absence of 10−8 M VIP or PACAP. The neuropeptides were removed by extensive washing at different times (from 5–180 min), and the cells were cultured in medium containing LPS without neuropeptides. Five-minute incubations resulted in significant stimulatory effects, and 15-min incubations led to maximum stimulation (Fig. 3 B).

FIGURE 3.

Time course for the stimulatory activity of VIP and PACAP. A, Macrophages were stimulated with LPS (0.5 μg/ml) at time zero, and VIP or PACAP (10−8 M) was added at different times after the initiation of the cultures. Supernatants were collected 24 h after the addition of the VIP/PACAP and assayed for IL-10. B, Peritoneal macrophages were stimulated with LPS (0.5 μg/ml) in the presence or the absence of VIP or PACAP (10−8 M). The neuropeptides were removed at different times by washing (three times with serum-free medium), and the cells were resuspended in complete medium containing LPS (0.5 μg/m) in the absence of neuropeptides and cultured for an additional 24 h. Control cultures containing only LPS were washed and recultured under the same conditions. Supernatants were collected, and the IL-10 content was determined by ELISA. The percent stimulation was calculated by comparing each experimental culture with its control. Each result is the mean ± SD of four experiments.

FIGURE 3.

Time course for the stimulatory activity of VIP and PACAP. A, Macrophages were stimulated with LPS (0.5 μg/ml) at time zero, and VIP or PACAP (10−8 M) was added at different times after the initiation of the cultures. Supernatants were collected 24 h after the addition of the VIP/PACAP and assayed for IL-10. B, Peritoneal macrophages were stimulated with LPS (0.5 μg/ml) in the presence or the absence of VIP or PACAP (10−8 M). The neuropeptides were removed at different times by washing (three times with serum-free medium), and the cells were resuspended in complete medium containing LPS (0.5 μg/m) in the absence of neuropeptides and cultured for an additional 24 h. Control cultures containing only LPS were washed and recultured under the same conditions. Supernatants were collected, and the IL-10 content was determined by ELISA. The percent stimulation was calculated by comparing each experimental culture with its control. Each result is the mean ± SD of four experiments.

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To study the second messengers involved in the effect of VIP and PACAP, we investigated the effects of calphostin C (a PKC inhibitor) (34), H89 (a PKA inhibitor) (35), and forskolin and PGE2 (two strict cAMP-inducing agents) (36) on the release of IL-10. Forskolin and PGE2 stimulated IL-10 production similar to VIP and PACAP (Fig. 4,A), suggesting the involvement of cAMP. The role of cAMP as second messenger is also supported by the effect of the PKA inhibitor H89. In the concentration range in which calphostin C (the PKC inhibitor) does not affect IL-10 production in LPS-stimulated cells (Fig. 4,B, right panel), it does not reverse the stimulatory effect of VIP/PACAP (Fig. 4,B, left panel). In contrast, H89 (the PKA inhibitor) reversed in a dose-dependent manner the stimulatory effect of VIP/PACAP (Fig. 4 B, center panel). These results suggest that the stimulatory effect of VIP/PACAP on IL-10 production is mediated through increases in intracellular cAMP.

FIGURE 4.

cAMP as a second messenger for the stimulatory activity of VIP and PACAP on IL-10 production. A, Effects of various cAMP-inducing agents. Peritoneal macrophages were stimulated with LPS (0.5 μg/ml) in the presence or the absence of different concentrations of VIP, PACAP, forskolin (FK), or PGE2. Twenty-four-hour culture supernatants were assayed for IL-10 by ELISA. Each result is the mean ± SD of five experiments. B, Comparative effects of calphostin C (a PKC inhibitor) and H89 (a PKA inhibitor) on the stimulatory activity of VIP and PACAP. Macrophages were stimulated with LPS (0.5 μg/ml), LPS plus VIP (10−8 M), or LPS plus PACAP (10−8 M) in the absence or the presence of different concentrations of calphostin C or H89. Twenty-four-hour culture supernatants were assayed for IL-10 by ELISA. The dotted line represents control values from cultures incubated with LPS alone (2978 ± 139 pg IL-10/ml). Each result is the mean ± SD of five experiments. ∗, p < 0.001 with respect to neuropeptide-treated samples without protein kinase inhibitors.

FIGURE 4.

cAMP as a second messenger for the stimulatory activity of VIP and PACAP on IL-10 production. A, Effects of various cAMP-inducing agents. Peritoneal macrophages were stimulated with LPS (0.5 μg/ml) in the presence or the absence of different concentrations of VIP, PACAP, forskolin (FK), or PGE2. Twenty-four-hour culture supernatants were assayed for IL-10 by ELISA. Each result is the mean ± SD of five experiments. B, Comparative effects of calphostin C (a PKC inhibitor) and H89 (a PKA inhibitor) on the stimulatory activity of VIP and PACAP. Macrophages were stimulated with LPS (0.5 μg/ml), LPS plus VIP (10−8 M), or LPS plus PACAP (10−8 M) in the absence or the presence of different concentrations of calphostin C or H89. Twenty-four-hour culture supernatants were assayed for IL-10 by ELISA. The dotted line represents control values from cultures incubated with LPS alone (2978 ± 139 pg IL-10/ml). Each result is the mean ± SD of five experiments. ∗, p < 0.001 with respect to neuropeptide-treated samples without protein kinase inhibitors.

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Having demonstrated that VIP and PACAP had a stimulatory effect on IL-10 production, we sought to determine whether this action occurred at a transcriptional level. We stimulated peritoneal macrophages with LPS in the presence or the absence of 10−8 M VIP or PACAP for 2, 4, 8, and 18 h, and total RNA was prepared and subjected to Northern blot analysis. Although no IL-10 mRNA was detectable in unstimulated cells (Fig. 5,A), time-dependent increasing levels of IL-10 mRNA are present in LPS-stimulated cells (Fig. 5, B and C). At all time points, VIP and PACAP significantly increased the levels of specific IL-10 mRNA (Fig. 5, B and C). These results indicate that both neuropeptides exert their actions at the level of mRNA.

FIGURE 5.

VIP and PACAP stimulate IL-10 transcription. A and B, Macrophages were stimulated with LPS (0.5 μg/ml) and incubated with or without VIP or PACAP (10−8 M) for 8 h (A) and for 2, 4, 8, and 18 h (B). The expression of IL-10 and β-actin mRNA was analyzed by Northern blot analysis at the indicated time points. C, Results are expressed in arbitrary densitometric units normalized for the expression of β-actin in each sample. One representative experiment of three is shown.

FIGURE 5.

VIP and PACAP stimulate IL-10 transcription. A and B, Macrophages were stimulated with LPS (0.5 μg/ml) and incubated with or without VIP or PACAP (10−8 M) for 8 h (A) and for 2, 4, 8, and 18 h (B). The expression of IL-10 and β-actin mRNA was analyzed by Northern blot analysis at the indicated time points. C, Results are expressed in arbitrary densitometric units normalized for the expression of β-actin in each sample. One representative experiment of three is shown.

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In addition, to confirm that VIP and PACAP stimulate the de novo synthesis of IL-10, LPS-stimulated macrophages were incubated with cycloheximide, a protein synthesis inhibitor, or with actinomycin D, a transcriptional inhibitor, in the presence or the absence of VIP or PACAP. Both cycloheximide and actinomycin D reduced IL-10 to undetectable levels (Fig. 6), suggesting that VIP and PACAP induce de novo transcription and synthesisof IL-10.

FIGURE 6.

Effects of cycloheximide and actinomycin D on the stimulatory activity of VIP and PACAP. Macrophages were stimulated with LPS (0.5 μg/ml), LPS plus VIP (10−8 M), or LPS plus PACAP (10−8 M) for 3.5 h before the addition of actinomycin D (ActD; 5 μg/ml) or for 8 h before the addition of cycloheximide (CHX; 1 μg/ml). Supernatants were collected 8 h later and assayed for IL-10 production by ELISA. Each result is the mean ± SD of four experiments.

FIGURE 6.

Effects of cycloheximide and actinomycin D on the stimulatory activity of VIP and PACAP. Macrophages were stimulated with LPS (0.5 μg/ml), LPS plus VIP (10−8 M), or LPS plus PACAP (10−8 M) for 3.5 h before the addition of actinomycin D (ActD; 5 μg/ml) or for 8 h before the addition of cycloheximide (CHX; 1 μg/ml). Supernatants were collected 8 h later and assayed for IL-10 production by ELISA. Each result is the mean ± SD of four experiments.

Close modal

Although the IL-10 promoter contains a complex array of transactivating binding sites, the cAMP-responsive element appears to be essential for maximal IL-10 transcription (27, 37). In addition, it has been described that cAMP-elevating drugs, such as PGE2 and dibutyryl cAMP, up-regulate IL-10 production by macrophages (37, 38, 39, 40). To investigate whether VIP/PACAP affect CRE binding, we used EMSAs. Stimulation of macrophages with LPS led to a time-dependent increase in CRE binding compared with that in unstimulated cells, and treatment with VIP and PACAP significantly increased this binding (Fig. 7,A). The binding specificity was confirmed using homologous (CRE) and nonhomologous (NF-κB) oligonucleotides as competitors (Fig. 7,A). Ab supershift experiments were performed to determine the composition of the CRE-binding factors. In VIP/PACAP-treated cells, the majority of the complex was supershifted by an anti-CREB Ab, whereas no supershift was observed using an anti-c-Jun Ab (Fig. 7 B), indicating the presence of CREB in the CRE-binding complexes.

FIGURE 7.

VIP and PACAP stimulate CREB binding through the VPAC1/cAMP pathway. A, Nuclear extracts were prepared from peritoneal macrophages incubated for 2, 4, and 8 h with LPS (0.5 μg/ml) in the presence or the absence of VIP or PACAP (10−8 M). CRE binding was assessed by EMSA using a radiolabeled oligonucleotide containing the murine CRE site of the IL-10 promoter. Specificity was determined by the addition of a 50-fold excess of unlabeled nonhomologous (NF-κB) or homologous (CRE) oligonucleotides to the nuclear extracts (Comp). B, Identification of the proteins bound to the CRE site using supershift analysis. Nuclear extracts (8-h incubation) were incubated with polyclonal Abs against CREB or c-Jun for 20 min before addition of the CRE probe. C, Nuclear extracts were prepared from macrophages incubated for 8 h with LPS (0.5 μg/ml; lane 1); LPS plus VIP (10−8 M; lane 2); LPS, VIP, and VPAC1 antagonist (10−6 M; lane 3); LPS, VIP, and H89 (100 nM; lane 4), or LPS plus forskolin (10−6 M; lane 5). The proteins bound to the CRE site were identified by supershift analysis. Similar results were observed in three independent experiments.

FIGURE 7.

VIP and PACAP stimulate CREB binding through the VPAC1/cAMP pathway. A, Nuclear extracts were prepared from peritoneal macrophages incubated for 2, 4, and 8 h with LPS (0.5 μg/ml) in the presence or the absence of VIP or PACAP (10−8 M). CRE binding was assessed by EMSA using a radiolabeled oligonucleotide containing the murine CRE site of the IL-10 promoter. Specificity was determined by the addition of a 50-fold excess of unlabeled nonhomologous (NF-κB) or homologous (CRE) oligonucleotides to the nuclear extracts (Comp). B, Identification of the proteins bound to the CRE site using supershift analysis. Nuclear extracts (8-h incubation) were incubated with polyclonal Abs against CREB or c-Jun for 20 min before addition of the CRE probe. C, Nuclear extracts were prepared from macrophages incubated for 8 h with LPS (0.5 μg/ml; lane 1); LPS plus VIP (10−8 M; lane 2); LPS, VIP, and VPAC1 antagonist (10−6 M; lane 3); LPS, VIP, and H89 (100 nM; lane 4), or LPS plus forskolin (10−6 M; lane 5). The proteins bound to the CRE site were identified by supershift analysis. Similar results were observed in three independent experiments.

Close modal

Since the stimulatory effect of VIP and PACAP on IL-10 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 CRE binding. The stimulatory activity of VIP on CRE binding was completely reversed by the VPAC1 antagonist and H89 (Fig. 7,C). These results suggest that the stimulation of CRE binding by VIP is mediated through VPAC1 and the subsequent elevation of cAMP levels. This is supported by the fact that forskolin affected CRE binding the same way as VIP (Fig. 7 C).

An attempt was made to reproduce the in vitro observations in vivo. The i.p. injection of LPS (100 μg) resulted in elevation of IL-10 in serum and peritoneal exudate fluid (Fig. 8,A). Treatment of mice with VIP or PACAP significantly enhanced the LPS-induced IL-10 level in serum and peritoneal fluid (Fig. 8,A). The in vivo effects of VIP and PACAP were dose dependent, with a maximum effect at 5–10 nmol/animal (Fig. 8,B). In addition, VIP and PACAP significantly stimulated IL-10 transcription in freshly isolated peritoneal exudate cells, 2 and 4 h after the LPS challenge (Fig. 8 C).

FIGURE 8.

In vivo effects of VIP and PACAP on LPS-induced IL-10 production. A, Mice (groups of three) were injected i.p. with LPS (100 μg/mouse), LPS plus VIP (5 nmol/mouse), or LPS plus PACAP (5 nmol/mouse). Serum and peritoneal fluid were obtained at the indicated time points as described in Materials and Methods and were assayed for IL-10 by ELISA. Each result is the mean ± SD of three experiments. B, Mice (groups of three) were injected i.p. with LPS (100 μg/mouse) in the presence or the absence of different concentrations of VIP or PACAP (0.5–10 nmol/mouse). Serum and peritoneal fluid were obtained after 8 h and assayed for IL-10 by ELISA. Each result is the mean ± SD of three experiments. C, Expression of IL-10 mRNA in peritoneal exudate cells. Mice (groups of three) were injected i.p. with medium alone (control for basal IL-10 mRNA), LPS (100 μg/mouse), LPS plus VIP (5 nmol/mouse), or LPS plus PACAP (5 nmol/mouse), and peritoneal exudate cells were harvested at 2 h (left panel) and at 2, 4, and 8 h (right panel). The expression of IL-10 mRNA was determined by Northern blot analysis. Results (lower panel) are expressed in arbitrary densitometric units normalized for the expression of β-actin in each sample. One representative experiment of three is shown.

FIGURE 8.

In vivo effects of VIP and PACAP on LPS-induced IL-10 production. A, Mice (groups of three) were injected i.p. with LPS (100 μg/mouse), LPS plus VIP (5 nmol/mouse), or LPS plus PACAP (5 nmol/mouse). Serum and peritoneal fluid were obtained at the indicated time points as described in Materials and Methods and were assayed for IL-10 by ELISA. Each result is the mean ± SD of three experiments. B, Mice (groups of three) were injected i.p. with LPS (100 μg/mouse) in the presence or the absence of different concentrations of VIP or PACAP (0.5–10 nmol/mouse). Serum and peritoneal fluid were obtained after 8 h and assayed for IL-10 by ELISA. Each result is the mean ± SD of three experiments. C, Expression of IL-10 mRNA in peritoneal exudate cells. Mice (groups of three) were injected i.p. with medium alone (control for basal IL-10 mRNA), LPS (100 μg/mouse), LPS plus VIP (5 nmol/mouse), or LPS plus PACAP (5 nmol/mouse), and peritoneal exudate cells were harvested at 2 h (left panel) and at 2, 4, and 8 h (right panel). The expression of IL-10 mRNA was determined by Northern blot analysis. Results (lower panel) are expressed in arbitrary densitometric units normalized for the expression of β-actin in each sample. One representative experiment of three is shown.

Close modal

VIP and PACAP are potent anti-inflammatory agents that down-regulate the activation of T cells and macrophages (7, 8, 41). Several other properties, especially vasodilation, make them of potential benefit in metabolically unfavorable circumstances such as hypoxia or ischemia (42). Recently, VIP and PACAP were shown to modulate the macrophage secretion of proinflammatory cytokines such as TNF-α and IL-6 (9, 11, 63). This might have clinical relevance, since these cytokines are involved in the detrimental effects of ischemia-reperfusion and septic shock (1, 43, 44). The present study shows a novel property of VIP/PACAP that might contribute to their anti-inflammatory effects, e.g., the stimulation of IL-10 production in LPS-activated macrophages. The stimulatory effect is dose dependent within a wide range of neuropeptide concentrations (10−6–10−10 M), with the maximum effect being observed at 10−8 M. This is the dose range in which VIP and PACAP modulate several other immunological functions (7, 8, 9, 41).

The time course indicates that, similar to the effect on IL-6 and TNF-α (25, 63), VIP and PACAP affect IL-10 production rapidly, with 5- to 15-min incubations sufficient to achieve maximum stimulatory effects. In addition, the stimulation has a refractory period of 12 h, after which the stimulatory activity of VIP/PACAP is reduced significantly.

Of relevance is the fact that in the absence of LPS, VIP and PACAP do not stimulate IL-10 release. This observation indicates that VIP/PACAP can only amplify an endotoxin-generated signal and suggests that the in vivo action of VIP/PACAP may be restricted to cells that are actively involved in responding to Ags.

Similar to the effect on other cytokines such as IL-2, IL-6, TNF-α, and IL-12 (7, 27, 63)4 the stimulation of IL-10 requires intact VIP/PACAP molecules. This is in agreement with previous reports showing that either C- or N-terminal truncations of VIP lead to significant losses in biological activity (45, 46). Peritoneal macrophages have been previously shown to express VPAC1 and PAC1 mRNA, and both high and low affinity VIP/PACAP binding sites (16, 17). In addition, we have recently reported that LPS-stimulated Raw 264.7 macrophages express mRNA for all three VIP/PACAP receptors (47). Our agonist studies suggest that VPAC1 mediates the stimulatory effect on IL-10. This is in agreement with Dewit et al. (11), who reported that a VPAC2 agonist has no effect on IL-10 production in human blood monocytes. The role of VPAC1 as the unique mediator in the effect on IL-10 production is also supported by the fact that a VPAC1 antagonist, but not PACAP6–38, an antagonist specific for PAC1 and to a lesser degree for VPAC2 (32), reverses the stimulatory effect of VIP/PACAP. Also, the VPAC1 antagonist blocked the effect of VIP/PACAP on CREB binding to the CRE site specific for the IL-10 promoter, supporting the involvement of VPAC1 in the stimulatory effect of VIP/PACAP on IL-10 gene expression.

The VPAC1 is coupled primarily to the adenylate cyclase system (14). To fully understand the mechanism of action of VIP and PACAP, it is important to clarify which transduction pathways are involved in the stimulation of IL-10 in macrophages. It has been reported that in monocytes/macrophages IL-10 production is stimulated by cAMP-inducing agents (37, 38, 39, 40). In the present study forskolin and PGE2, two strict cAMP-inducing agents (36), stimulate IL-10 release similar to VIP and PACAP. In addition, H89, a potent and selective PKA inhibitor (35), completely reverses the effect of VIP/PACAP. In contrast, calphostin C (a specific PKC inhibitor) (34) does not alter the stimulatory action of either VIP or PACAP. These results suggest that VIP/PACAP stimulate IL-10 production in macrophages through the cAMP/PKA pathway initiated through binding to VPAC1.

Previous experiments regarding VIP modulation of cytokine expression indicated different molecular mechanisms, i.e., transcriptional regulation for IL-2, IL-6, and TNF-α vs post-transcriptional regulation for IL-4 (9, 47, 48, 63). The present study indicates that the stimulatory effect of VIP and PACAP on IL-10 production occurs at the mRNA level. This is supported by the fact that VIP/PACAP induce increases in steady state IL-10 mRNA, and that the stimulatory effect on the IL-10 release is inhibited by both transcriptional and protein synthesis inhibitors. It remains to be established whether the augmentation of steady state IL-10 mRNA levels results from an increase in either or both the novo transcriptional rate and message stabilization.

Although the IL-10 promoter contains a complex array of transactivating binding sites, the CRE appears essential for maximal IL-10 transcription in monocytes/macrophages (27, 37). Since VIP and forskolin induce similar increases in CRE-binding nuclear proteins, and H89 reverses the effect of VIP on CRE binding, we propose that VIP/PACAP stimulate IL-10 gene expression in peritoneal macrophages through a cAMP-dependent increase in the functionally active transcriptional factor CREB. In this respect, VIP and PACAP were reported recently to increase CREB phosphorylation and CREB-regulated transcription in several cell types (49, 50, 51). Since VIP/PACAP do not promote IL-10 production in the absence of LPS, the sole increase in CRE-binding activity is probably insufficient to induce the expression of the IL-10 gene. However, CREB could act in coordination with other transcriptional factors regulated by LPS, to induce maximal transcriptional activity for the IL-10 gene.

The necessity of several transcriptional factors acting in concert for the expression of the IL-10 gene may also explain the differences in the effects of VIP on IL-10 production in activated T cells vs activated macrophages. Previously we reported that VIP inhibits IL-10 expression in anti-CD3-stimulated T cells (26). This is in contrast to the present study, which indicates that VIP/PACAP stimulate IL-10 production in LPS-activated macrophages. In both cases, forskolin mimicked the effect of VIP, suggesting that the major transduction pathway was cAMP dependent. However, the nature of second messengers downstream from cAMP might be different in T cells and macrophages. Also, whereas in macrophages CREB induced by VIP/PACAP might act in concert with transcriptional factors induced by LPS, in T cells cAMP-dependent events such as the inhibition of JNK (52) might lead to the reduction in functionally active c-Jun protein and subsequent reduction in the transcriptional activity at the AP-1 site. Such events have been identified for the IL-2 promoter in anti-CD3-stimulated T cells treated with VIP (our unpublished observations).

The in vitro stimulatory effect of VIP/PACAP on IL-10 production correlates with the in vivo stimulation of both IL-10 expression and release in endotoxemic mice. VIP and PACAP both accelerate and increase the levels of circulating IL-10 and the IL-10 mRNA expression in peritoneal cells in endotoxemic mice. In contrast to the effect on IL-10, VIP and PACAP inhibit both TNF-α and IL-6 expression in endotoxemic mice (9, 12, 63). Therefore, through the inhibition of proinflammatory cytokines such as TNF-α and IL-6 and the stimulation of anti-inflammatory cytokines such as IL-10, VIP/PACAP released within the lymphoid microenvironment may play an important role in the down-regulation of the inflammatory response by significantly affecting the local balance between pro- and anti-inflammatory factors. The stimulation of the IL-10 transcription by VIP/PACAP may also have a significant therapeutical potential, since the in vivo administration of this anti-inflammatory cytokine leads to the prevention or reduction of a variety of inflammatory diseases such as endotoxic shock (53, 54, 55), viral-induced ocular inflammation (56), immune complex-mediated lung injury (57), and chronic inflammatory bowel disease (58).

Although VIP-ergic nerve fibers are found in most lymphoid organs, the respiratory and gastrointestinal tracts are particularly rich in VIP-containing fibers and cells (41, 59, 60), which release VIP upon electrical or chemical stimulation. Nitric oxide, for example, functions as a particularly potent signal for VIP release from enteric ganglia (61, 62). Therefore, nitric oxide produced during an inflammatory response could induce the release of high levels of VIP locally, especially in the respiratory and gastrointestinal tract, and the released neuropeptides could target the alveolar or peritoneal macrophages found in the immediate vicinity and down-regulate the production of proinflammatory agents.

We thank Dr. Patrick Robberecht (Universite Libre de Bruxelles, Brussels, Belgium) for the VPAC1 agonist and antagonist, Drs. David Bolin and Ann Welton (Hoffmann-La Roche, Nutley, NJ) for the VPAC2 agonist Ro 25–1553, and Dr. Ethan Lerner (Massachusetts General Hospital, Charlestown, MA) for the PAC1 agonist maxadilan.

1

This work was supported by Public Health Service Grant AI041786-01 (to D.G.), Busch Biomedical Award 98-00 (to D.G.), Grant PB94-0310 (to R.P.G.), and a postdoctoral fellowship from the Spanish Department of Education and Science (PM98-0081 to M.D.).

3

Abbreviations used in this paper: VIP, vasoactive intestinal peptide; PACAP, pituitary adenylate cyclase-activating polypeptide; VPAC1, type 1 VIP receptor; VPAC2, type 2 VIP receptor; GRF, growth hormone-releasing factor; H89, N-[2-(p-bromocinnamyl-amino)ethyl]-5-iso-quinolinesulfonamide; EMSA, electrophoretic mobility shift assay; CRE, adenosine 3′,5′-monophosphate-responsive element; CREB, adenosine 3′,5′-monophosphate-responsive element-binding protein; PAC1, pituitary adenylate cyclase-activating polypeptide receptor; PKC, protein kinase C; PKA, protein kinase A.

4

Delgado, M., E. J. Munoz-Elias, R. P. Gomariz, and D. Ganea. VIP and PACAP inhibit IL-12 production in LPS-stimulated macrophages: subsequent effect on T cell IFN-γ synthesis. Submitted for publication.

1
Laskin, D. L., K. J. Pendino.
1995
. Macrophages and inflammatory mediators in tissue injury.
Annu. Rev. Pharmacol. Toxicol.
35
:
655
2
Kunkel, S. L., M. Spengler, M. A. May, R. Spengler, J. Larrick, D. Remick.
1988
. Prostaglandin E2 regulates macrophage-derived tumor necrosis factor gene expression.
J. Biol. Chem.
263
:
5380
3
Trepicchio, W., M. Bozza, G. Pedneault, A. Dorner.
1996
. Recombinant human IL-11 attenuates the inflammatory response through down-regulation of proinflammatory cytokine release and nitric oxide production.
J. Immunol.
157
:
3627
4
Fiorentino, D. F., A. Zlotnik, T. R. Mossman, M. Howard, A. O’Garra.
1991
. Interleukin-10 inhibits cytokine production by activated macrophages.
J. Immunol.
147
:
3815
5
Tsunawaki, S., M. Sporn, A. Ding, C. F. Nathan.
1988
. Deactivation of macrophages by transforming growth factor β.
Nature
334
:
260
6
Muchamuel, T., S. Menon, P. Pisacane, M. C. Howard, D. A. Cockayne.
1997
. IL-13 protects mice from LPS-induced lethal endotoxemia.
J. Immunol.
158
:
2898
7
Ganea, D..
1996
. Regulatory effects of vasoactive intestinal peptide on cytokine production in central and peripheral lymphoid organs.
Adv. Neuroimmunol.
6
:
61
8
De la Fuente, M., M. Delgado, R. P. Gomariz.
1996
. VIP modulation of immune cell functions.
Adv. Neuroimmunol.
6
:
75
9
Martinez, C., M. Delgado, D. Pozo, J. Leceta, J. R. Calvo, D. Ganea, R. P. Gomariz.
1998
. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide modulate endotoxin-induced IL-6 production by murine peritoneal macrophages.
J. Leukocyte Biol.
63
:
591
10
Hernanz, A., E. Tato, M. De la Fuente, E. De Miguel, F. Arnalich.
1996
. Differential effects of gastrin-releasing peptide, neuropeptide Y, somatostatin and vasoactive intestinal peptide on interleukin-1β, interleukin-6 and tumor necrosis factor-α production by whole blood cells from healthy young and old subjects.
J. Neuroimmunol.
71
:
25
11
Dewit, D., P. Gourlet, Z. Amraoui, P. Vertongen, F. Willems, P. Robberecht, M. Goldman.
1998
. The vasoactive intestinal peptide analogue RO25–1553 inhibits the production of TNF and IL-12 by LPS-activated monocytes.
Immunol. Lett.
60
:
57
12
Delgado, M., C. Martinez, D. Pozo, J. R. Calvo, J. Leceta, D. Ganea, and R. P. Gomariz. 1999. VIP and PACAP protect mice from lethal endotoxemia through the inhibition of TNF-α and IL-6. J. Immunol. In press.
13
Harmar, A. J., A. Arimura, I. Gozes, L. Journot, M. Laburthe, J. R. Pisegna, S. R. Rawlings, P. Robberecht, S. I. Said, S. P. Sreedharan, et al
1999
. Nomenclature of receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP).
Pharmacol. Rev.
50
:
625
14
Rawlings, S. R., M. Hezareh.
1996
. Pituitary adenylate cyclase activating polypeptide and PACAP/vasoactive intestinal polypeptide receptors: actions on the anterior pituitary gland.
Endocr. Rev.
17
:
4
15
Segura, J. J., J. M. Guerrero, R. Goberna, J. R. Calvo.
1991
. Characterization of functional receptors for vasoactive intestinal peptide (VIP) in rat peritoneal macrophages.
Regul. Pept.
33
:
133
16
Delgado, M., D. Pozo, C. Martinez, E. Garrido, J. Leceta, J. R. Calvo, R. P. Gomariz.
1996
. Characterization of gene expression of VIP and VIP1-receptor in rat peritoneal lymphocytes and macrophages.
Regul. Pept.
62
:
161
17
Pozo, D., M. Delgado, C. Martinez, R. P. Gomariz, J. M. Guerrero, J. R. Calvo.
1997
. Functional characterization and mRNA expression of pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor in rat peritoneal macrophages.
Biochim. Biophys. Acta
1359
:
250
18
Fiorentino, D. F., M. W. Bond, T. R. Mosmann.
1989
. Two types of mouse T helper cell. IV. TH2 clones secrete a factor that inhibits cytokine production by TH1 clones.
J. Exp. Med.
170
:
2081
19
de Waal Malefyt, R., J. Haanen, H. Spits, M. G. Roncarolo, A. te Velde, C. Figdor, K. Johnson, R. Kastelein, H. Yssel, J. E. de Vries.
1991
. IL-10 and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II histocompatibility complex expression.
J. Exp. Med.
174
:
915
20
de Waal Malefyt, R., J. Abrams, B. Bennett, C. G. Figdor, J. E. de Vries.
1991
. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes.
J. Exp. Med.
174
:
1209
21
Barsig, J., S. Kusters, K. Vogt, H. D. Volk, G. Tiegs, A. Wendel.
1995
. Lipopolysaccharide-induced interleukin-10 in mice: role of endogenous tumor necrosis factor-α.
Eur. J. Immunol.
25
:
2888
22
Bogdan, C., Y. Vodovotz, C. Nathan.
1991
. Macrophage deactivation by interleukin 10.
J. Exp. Med.
174
:
1549
23
D’Andrea, A., M. Aste-Amezaga, N. M. Valiante, X. Ma, M. Kubin, G. Trinchieri.
1993
. Interleukin 10 inhibits human IFNγ production by suppressing natural killer cell stimulatory factor IL-12 synthesis in accessory cells.
J. Exp. Med.
178
:
1041
24
Wang, P., P. Wu, M. I. Siegel, R. W. Egan, M. M. Bilah.
1994
. IL-10 inhibits transcription of cytokine genes in human peripheral blood mononuclear cells.
J. Immunol.
153
:
811
25
Aste-Amezaga, M., X. Ma, A. Sartoiri, G. Trinchieri.
1998
. Molecular mechanisms of the induction of IL-12 and its inhibition by IL-10.
J. Immunol.
160
:
5936
26
Martinez, C., M. Delgado, R. P. Gomariz, D. Ganea.
1996
. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide-38 inhibit IL-10 production in murine T lymphocytes.
J. Immunol.
156
:
4128
27
Kim, J. M., C. I. Brannan, N. G. Copeland, N. A. Jenkins, T. A. Khan, K. W. Moore.
1992
. Structure of the mouse IL-10 gene and chromosomal localization of the mouse and human genes.
J. Immunol.
148
:
3618
28
O’Garra, A., R. Chang, N. Go, R. Hastings, G. Haughton, M. Howard.
1992
. Ly-1 B (B-1) cells are the main source of B-cell derived interleukin-10.
Eur. J. Immunol.
22
:
711
29
Gourlet, P., A. Vandermeers, P. Vertongen, J. Ratche, P. De Neef, J. Cnudde, M. Waelbroeck, and P. Robberecht. Development of high affinity selective VIP1 receptor agonists. Peptides 18:1539–1545.
30
Gourlet, P., P. Vertongen, A. Vandermeers, M. C. Vandermmers-Piret, J. Rathe, P. De Neef, M. Waelbroeck, P. Robberecht.
1997
. The long-acting vasoactive intestinal polypeptide agonist RO25-1553 is highly selective of the VIP2 receptor subclass.
Peptides
18
:
403
31
Moro, O., E. A. Lerner.
1996
. Maxadilan, the vasodilator peptide from sand flies, is a specific pituitary adenylate cyclase activating peptide type I receptor agonist.
J. Biol. Chem.
271
:
966
32
Gourlet, P., M. C. Vandermeers-Piret, J. Rathe, P. De Neef, P. Robberecht.
1995
. Fragments of the pituitary adenylate cyclase-activating polypeptide discriminate between type I and II recombinant receptors.
Eur. J. Pharmacol.
287
:
7
33
Gourlet, P., P. De Neef, J. Cnudde, M. Waelbroeck, P. Robberecht.
1997
. In vitro properties of a high affinity selective antagonist of the VIP1 receptor.
Peptides
18
:
1555
34
Kobayashi, E., H. Nakno, M. Morimoto, T. Tamaoki.
1989
. Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C.
Biochem. Biophys. Res. Commun.
159
:
548
35
Chijiwa, T., A. Mishima, M. Hagiwara, M. Sano, K. Hayashi, T. Inoue, K. Naito, T. Toshioka, H. Hidaka.
1990
. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromociannamylamino)ethyl]-5-isoquinoleinsulfonamide (H89), of PC12D pheochromocytoma cells.
J. Biol. Chem.
265
:
5267
36
Boige, N., B. Aminaroff, A. Munk, M. Laburthe.
1984
. Forskolin stimulates adenylate cyclase in human colonic crypts: interaction with VIP.
Eur. J. Pharmacol.
101
:
111
37
Platzer, C., C. Meisel, K. Vogt, M. Platzer, H. D. Volk.
1995
. Up-regulation of monocytic IL-10 by tumor necrosis factor-α and cAMP elevating drugs.
Int. Immunol.
7
:
517
38
Strassmann, G., V. Patil-Koota, F. Finkelman, M. Fong, T. Kambayashi.
1995
. Evidence for the involvement of interleukin-10 in the differential deactivation of murine peritoneal macrophages by prostaglandin E2.
J. Exp. Med.
180
:
2365
39
Arai, T., K. Hiromatsu, N. Kobayashi, M. Takano, H. Ishida, Y. Nimura, Y. Yoshikai.
1995
. IL-10 is involved in the protective effect of dibutyryl cyclic adenosine monophosphate on endotoxin-induced inflammatory liver injury.
J. Immunol.
155
:
5743
40
Kambayashi, T., C. O. Jacob, D. Zhou, N. Mazurek, M. Fong, G. Strassmann.
1995
. Cyclic nucleotide phosphodiesterase type IV participates in the regulation of IL-10 and in the subsequent inhibition of TNF-α and IL-6 release by endotoxin-stimulated macrophages.
J. Immunol.
155
:
4909
41
Bellinger, D. L., D. Lorton, S. Brouxhon, S. Felten, D. L. Felten.
1996
. The significance of vasoactive intestinal peptide (VIP) in immunomodulation.
Adv. Neuroimmunol.
6
:
5
42
Said, S. I..
1985
. Vasoactive intestinal peptide. D. Raenburn, and M. A. Giembycz, eds.
Airways Smooth Muscle: Peptide Receptors, Ion Channels, and Signal Transduction
87
Birkhauser Verlag Basel, Switzerland.
43
Van Snick, J..
1990
. Interleukin 6: an overview.
Annu. Rev. Immunol.
8
:
253
44
Vassali, P..
1992
. The pathophysiology of tumor necrosis factor.
Annu. Rev. Immunol.
10
:
411
45
Gourlet, P., J.-P. Vilardaga, P. De Neef, M. Waelbroeck, A. Vandermeers, P. Robberecht.
1996
. The C-terminus ends of secretin and VIP interact with the N-terminal domains of their receptors.
Peptides
17
:
825
46
O’Donnell, M., R. J. Garippa, N. C. O’Neill, D. R. Bolin, J. M. Cottrell.
1991
. Structure-activity studies of vasoactive intestinal polypeptide.
J. Biol. Chem.
266
:
6389
47
Delgado, M., E. J. Munoz-Elias, Y. Kan, I. Gozes, M. Fridkin, D. E. Brenneman, R. P. Gomariz, D. Ganea.
1998
. Vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide inhibit TNFα transcriptional activation by regulating NF-κB and CREB/c-Jun.
J. Biol. Chem.
273
:
31427
48
Wang, H.-Y., Z. Xin., H. Tang, D. Ganea.
1996
. Vasoactive intestinal peptide inhibits IL-4 production in murine T cells by a post-transcriptional mechanism.
J. Immunol.
156
:
3243
49
Muller, A., B. Lutz-Bucher, P. Kienlen-Campard, B. Koch, J. P. Loeffler.
1998
. Continuous activation of pituitary adenylate cyclase-activating polypeptide receptors elicits antipodal effects on cyclic AMP and inositol phospholipid signaling pathways in CATH.a cells: role of protein synthesis and protein kinases.
J. Neurochem.
70
:
1431
50
Aoki, Y., Y. Iwasaki, M. Katahira, Y. Oiso, H. Saito.
1997
. Regulation of the rat proopiomelanocortin gene expression in AtT-20 cells. II. Effects of the pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide.
Endocrinology
138
:
1930
51
Schomerus, C., E. Maronde, E. Laedtke, H. W. Korf.
1996
. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) induce phosphorylation of the transcription factor CREB in subpopulations of rat pinealocytes: immunocytochemical and immunochemical evidence.
Cell. Tissue Res.
286
:
305
52
Hsueh, Y.-P., M.-Z. Lai.
1995
. C-Jun N-terminal kinase but not mitogen-activated protein kinase is sensitive to cAMP inhibition in T lymphocytes.
J. Biol. Chem.
270
:
18094
53
Howard, M., T. Muchamuel, S. Andrade, S. Menon.
1993
. Interleukin 10 protects mice from lethal endotoxemia.
J. Exp. Med.
177
:
1205
54
Wang, R., Q. Fang, L. Zhang, L. Radvany, A. Sharma, N. Noben-Trauth, G. B. Mills, Y. Shi.
1997
. CD28 ligation prevents bacterial toxin-induced septic shock in mice by inducing IL-10 expression.
J. Immunol.
158
:
2856
55
van der Poll, T., P. M. Jansen, W. J. Montegut, C. C. Braxton, S. E. Calvano, S. A. Stackpole, S. R. Smith, S. W. Swanson, C. E. Hack, S. F. Lowry, et al
1997
. Effects of IL-10 on systemic inflammatory responses during sublethal primate endotoxemia.
J. Immunol.
158
:
1971
56
Tumpey, T. M., V. M. Elner, S. Chen, J. E. Oakes, R. N. Lausch.
1994
. Interleukin 10 treatment can suppress stromal keratitis induced by herpes simplex virus type 1.
J. Immunol.
153
:
2258
57
Mulligan, M. S., M. L. Jones, A. A. Vaporciyan, M. C. Howard, P. A. Wood.
1993
. Protective effects of IL-4, and IL-10 against immune complex induced lung injury.
J. Immunol.
151
:
5666
58
Schreiber, S., T. Heinig, H. G. Thiele, A. Raedler.
1995
. Immunoregulatory role of interleukin 10 in patients with inflammatory bowel disease.
Gastroenterology
108
:
1434
59
Pearse, A. G. E., J. M. Polak, S. R. Bloom.
1977
. The newer gut hormone: cellular sources, physiology, pathology, and clinical aspects.
Gastroenterology
72
:
746
60
Dey, R. D., W. A. Shannon, S. I. Said.
1981
. Localization of VIP-immunoreactive nerves in airways and pulmonary vessels of dogs, cats, and human subjects.
Cell Tissue Res.
220
:
231
61
Grider, J. R., J.-G. Jin.
1993
. Vasoactive intestinal peptide release and l-citrulline production from isolated ganglia of the myenteric plexus: evidence for regulation of vasoactive intestinal peptide release by nitric oxide.
Neuroscience
54
:
521
62
Allescher, H. D., M. Kurjak, A. Huber, P. Trudrung, V. Schusdziarra.
1996
. Regulation of VIP release from rat enteric nerve terminals: evidence for a stimulatory effect of NO.
Am. J. Physiol.
271
:
G568
63
Delgodo, M., D. Pozo, C. Martinez, J. Leceta, J. R. Calvo, D. Genea, and R. P. Gomariz. 1999. Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit endotoxin-induced TNF-α production by macrophages: in vitro and in vivo studies. J. Immunol. 162, In press.