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The Journal of Immunology, 2000, 165: 3051-3057.
Copyright © 00 by The American Association of Immunologists

Inhibition of IFN-{gamma}-Induced Janus Kinase-1-STAT1 Activation in Macrophages by Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide

Mario Delgado*,{dagger} and Doina Ganea*

* Department of Biological Sciences, Rutgers University, Newark, NJ 07102; and {dagger} Departamento Biologia Celular, Facultad de Biologia, Universidad Complutense, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two immunomodulatory neuropeptides that affect both innate and acquired immunity, down-regulate IL-12 p40 and inducible NO synthase expression in LPS/IFN-{gamma}-stimulated macrophages. We showed previously that VIP/PACAP inhibit NF-{kappa}B nuclear translocation through the stabilization of I{kappa}B and reduce IFN regulatory factor-1 (IRF-1) binding to the regulatory elements found in the IL-12 p40 and inducible NO synthase promoters. In this paper we studied the molecular mechanisms involved in the VIP/PACAP regulation of IRF-1 transactivating activity. Our studies indicate that the inhibition in IRF-1 binding correlates with a reduction in IRF-1 protein and mRNA in IFN-{gamma}-treated Raw 264.7 macrophages. In agreement with the described Janus kinase (Jak)1/Jak2/STAT1/IRF-1 activation pathway, VIP/PACAP inhibit Jak1/Jak2, STAT1 phosphorylation, and the binding of STAT1 to the GAS sequence motif in the IRF-1 promoter. The effects of VIP/PACAP are mediated through the specific VIP/PACAP receptor-1 and the cAMP/protein kinase A (PKA) transduction pathway, but not through the induction of suppressor of cytokine signaling-1 or suppressor of cytokine signaling-3. Because IFN-{gamma} is a major stimulator of innate immune responses in vivo, the down-regulation of IFN-{gamma}-induced gene expression by VIP and PACAP could represent a significant element in the regulation of the inflammatory response by endogenous neuropeptides.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The generation of an immune response involves the activation of effector cells and the subsequent production of cytokines, chemokines, and reactive oxygen and nitrogen intermediates. The activated macrophages are widely recognized as cells that play a direct role in inflammatory processes, as well as in the initiation, maintenance, and control of specific immune responses. These capabilities are in a dynamic flux controlled by complex combinations of stimuli received from the tissue microenvironment. In response to LPS and other activating agents, macrophages secrete NO and proinflammatory cytokines, such as TNF-{alpha}, IL-1ß, IL-6, and IL-12, and immunomodulatory cytokines such as TGFß1 and IL-10 (1).

IFN-{gamma} constitutes one of the most potent macrophage-activating factors. Binding of IFN-{gamma} to its receptor induces the assembly of an active receptor complex and consequent transphosphorylation of the receptor-associated Janus tyrosine kinases (Jak)3 Jak1 and Jak2 (2, 3). The activation of these kinases induces phosphorylation of the cytoplasmic tail of the receptor itself, which lacks intrinsic kinase activity. The cytosolic protein STAT1 is then recruited to the activated IFN-{gamma}-receptor complex and phosphorylated (2). Upon phosphorylation, STAT1 form homodimers and translocate to the nucleus where they bind to the IFN-{gamma}-activated site (GAS), also termed STAT binding element, found in the promoter of many IFN-{gamma}-induced genes including the IFN regulatory factor-1 (IRF-1) and ICAM-1 genes (4, 5, 6, 7). Many of the regulatory effects of IFN-{gamma} in macrophages appear to be mediated by IRF-1, which transactivates multiple effector genes including IL-12 and the inducible NO synthase (iNOS; Ref. 8, 9, 10, 11, 12, 13, 14, 15).

Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP) are two neuropeptides that perform a broad spectrum of biological functions affecting both natural and acquired immunity (reviewed in Refs. 16, 17, 18), primarily as anti-inflammatory agents. VIP and PACAP have been shown to inhibit T cell proliferation and cytokine production (reviewed in Ref. 19), and to modulate several macrophage functions, including phagocytosis, respiratory burst, and chemotaxis (reviewed in Ref. 17). In agreement with their anti-inflammatory role, VIP and PACAP were recently reported to inhibit the in vitro and in vivo production of proinflammatory cytokines such as IL-6 and TNF-{alpha} (20, 21, 22), to reduce the secretion of NO (23), to enhance the production of the anti-inflammatory cytokine IL-10 (24), to protect mice from endotoxic shock presumably through the inhibition of endogenous TNF-{alpha} and of other inflammatory mediators (25), and to act as survival factors against tissue injury of lung and neuronal cells (26, 27, 28). Furthermore, we and others have recently demonstrated that VIP and PACAP inhibit IL-12 production in endotoxin-stimulated peritoneal macrophages (29, 30, 31), with a subsequent inhibitory effect on IFN-{gamma} synthesis by T cells (31). Both the inhibitory and the stimulatory effects are exerted at a transcriptional level, and involve regulation of several transcription factors, such as NF-{kappa}B, cAMP response element binding protein, c-Jun, and IRF-1 (23, 24, 32, 33). In particular, VIP and PACAP inhibit IL-12 and iNOS gene expression stimulated by LPS and IFN-{gamma} partly through the inhibition of IRF-1 synthesis (23, 33). To further understand the molecular mechanisms through which VIP and PACAP attenuate the inflammatory responses, we examined the effects of VIP/PACAP on IFN-{gamma}-induced Jak/STAT1 activation and IRF-1 synthesis in Raw 264.7 macrophages. Our results indicate that VIP/PACAP inhibit Jak1/Jak2/STAT1 phosphorylation, binding of STAT1 to the GAS motif in the IRF-1 promoter, and subsequently, IRF-1 transcription and synthesis. These effects are mediated through the VIP/PACAP receptor (VPAC)1 and the cAMP/PKA transduction pathway. VIP/PACAP do not induce suppressor of cytokine signaling (SOCS)1/3 in nonstimulated macrophages or in LPS- and LPS/IFN-{gamma}-activated macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Synthetic VIP and PACAP38 were purchased from Novabiochem (Laufelfingen, Switzerland). The VPAC1 antagonist (Ac-His1, D-Phe2, K15, R16, L27) VIP (3, 4, 5, 6, 7)-GRF (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) was kindly donated by Dr. Patrick Robberecht (Universite Libre de Bruxelles, Brussels, Belgium). Oligonucleotides were synthesized by the Oligonucleotide Synthesis Service from Rutgers University (Newark, NJ). Murine recombinant IFN-{gamma} was purchased from PharMingen (San Diego, CA). Forskolin, protease inhibitors, PMSF, EDTA, glycine, protein G-Sepharose, glycerol, EGTA, and DTT were purchased from Sigma (St. Louis, MO), and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Abs against IRF-1, phospho-Tyr (PY20), phosphorylated-STAT1 (Tyr701), Jak1, Jak2, STAT1{alpha} p91, and NF-{kappa}B p65 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell cultures

Raw 264.7 mouse macrophage cells (American Type Culture Collection, Rockville, MD) were cultured in DMEM supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 10 µg/ml streptomycin, and 10% FCS (complete culture medium; Life Technologies, Rockville, MD). The cells were plated in flat-bottom 96-well microtiter plates (Corning Glass, Corning, NY) for 24 h. Nonadherent cells were removed by aspiration and two washings with DMEM medium. Peritoneal macrophages were obtained from male BALB/c mice (6 to 10-wk-old) previously injected i.p. with 4% Brewer’s thioglycolate medium (Difco, Detroit, MI). Peritoneal cells were collected 4 days later by peritoneal lavage with ice-cold DMEM. The peritoneal cells were washed twice with ice-cold DMEM and incubated for 2 h at 37°C. The nonadherent cells were removed by washing. At least 96% of the adherent cells were macrophages as judged by morphologic and phagocytic criteria, and FACS analysis (>96% Mac1+). Macrophage monolayers were incubated with complete medium and stimulated with 100 U/ml IFN-{gamma} in the presence or absence of VIP or PACAP38 (from 10-12 to 10-7 M) for various times at 37°C in a humidified incubator with 5% CO2.

RNA extraction and Northern blot analysis

Northern blot analysis was performed according to standard methods. Macrophage monolayers (2 x 106 cells/ml) were stimulated with 100 U/ml IFN-{gamma} in the absence or presence of 10-8 M VIP and PACAP 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 Schleicher & Schuell Nytran membranes (Keene, NJ), and cross-linked to the nylon membrane using UV light.

The probes for murine IRF-1 and GAPDH were generated by RT-PCR as previously described (34). The probes for SOCS1 and SOCS3 were generated by RT-PCR as previously described (35). Oligonucleotides were end-labeled with 3000 Ci/mmol [{gamma}-32P]ATP (Amersham, Arlington, IL) by using T4 polynucleotide kinase. The RNA-containing membranes were prehybridized for 16 h at 42°C, and hybridized at 42°C for 16 h with the appropriate probes. The membranes were washed twice in 2x SSC containing 0.1% SDS at room temperature for 20 min each time, once at 37°C for 20 min, and once in 0.1x SSC containing 0.1% SDS at 50°C for 20 min. The prehybridization and hybridization buffers were purchased from 5 Prime->3 Prime (Boulder, CO). The membranes were exposed to x-ray films (Kodak, Rochester, NY).

EMSA

Nuclear extracts were prepared by the mini-extraction procedure of Schreiber et al. (36) with slight modifications. Raw 264.7 cells were plated at a density of 107 cells in six-well plates, stimulated, washed twice with ice-cold PBS/0.1% BSA, and scraped off the dishes. The cell pellets were homogenized with 0.4 ml of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM NaN3). After 15 min on ice, Nonidet P-40 was added to a final 0.5% concentration, the tubes were gently vortexed for 15 s and nuclei were sedimented and separated from cytosol by centrifugation at 12,000 x g for 40 s. Pelleted nuclei were washed once with 0.2 ml of ice-cold buffer A, and the soluble nuclear proteins were released by adding 0.1 ml of buffer C (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM NaN3). After incubation for 30 min on ice, followed by centrifugation for 10 min at 14,000 rpm at 4°C, the supernatants containing the nuclear proteins were harvested, the protein concentration was determined by the Bradford method, and aliquots were stored at -80°C for later use in EMSAs.

Oligonucleotides corresponding to the IRF-1 (nucleotides -933 to -906) motif of the iNOS promoter and GAS site of IRF-1 promoter were synthesized as follows (nucleotides -131 to -105; Ref. 4, 12): 5'-CACTGTCAATATTTCACTTTCATAATG-3' (IRF-1) and 5'-GCCTGATTTCCCCGAAATGACGGC-3' (GAS). The oligonucleotides were annealed after incubation for 5 min at 85°C in 10 mM Tris-HCl (pH 8.0), 5 mM NaCl, 10 mM MgCl2 and 1 mM DTT. Aliquots of 50 ng of the double-stranded oligonucleotides were end-labeled with [{gamma}-32P]ATP by using T4 polynucleotide kinase. For EMSA we used 20,000–50,000 cpm of double-stranded oligonucleotides, corresponding to ~0.5 ng per reaction. The 15 µl binding reaction mixtures were set up containing the following: 0.5–1 ng DNA probe, 5 µg nuclear extract, 2 µg poly(dI-dC)·poly(dI-dC) and binding buffer (50 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 5% glycerol, and 10 mM Tris-HCl (pH7.5)). The mixtures were incubated on ice for 15 min before adding the probe, followed by another incubation for 20 min at room temperature. Samples were loaded onto 4% nondenaturing polyacrylamide gels and electrophoresed in TGE buffer (50 mM Tris-HCl (pH7.5), 0.38 M glycine, and 2 mM EDTA) at 100 V, followed by transfer to Whatman paper, drying under vacuum at 80°C, and autoradiography. In competition and Ab supershift experiments, the nuclear extracts were incubated for 15 min at room temperature with the specific Ab (1 µg) or competing cold oligonucleotide (50-fold excess) before the addition of the labeled probe.

Western blot analysis of IRF-1

Thirty micrograms of nuclear extract protein was separated by SDS-PAGE (10% acrylamide). After electrophoresis, the gel was electroblotted in Tris-glycine buffer (48 mM Tris, 39 mM glycine; pH 9.2) containing 40% methanol onto a reinforced nitrocellulose membrane (Schleicher & Schuell). The membrane was blocked with TBST buffer (10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Tween 20) containing 5% milk powder for 1 h at room temperature, then incubated for an additional 6 h at 4°C with a rabbit polyclonal Ab against IRF-1 (1:500) in 5% nonfat milk/TBST solution. After washing three times in TBST, membranes were incubated at room temperature for 1 h with peroxidase-conjugated goat anti-rabbit IgG at 1:5000 dilution. After washing three times in TBST for 5 min each, and once in TBS for 5 min, the membrane was drained briefly and subjected to the enhanced chemiluminescence detection system (Amersham). The x-ray films were exposed for 5–20 min.

Analysis of STAT1 and Jak1/2 phosphorylation

The phosphorylation status of Jak1/Jak2 and STAT1 was assessed by immunoprecipitation followed by immunoblot analysis as previously described (37). Cells (3 x 107 cells) were lysed in ice-cold immunoprecipitation buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA (pH 7.4), 1 mM Na3VO4, 20 mM ß-glycerol phosphate, 1 mM PMSF, 1 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin, 1% Nonidet P-40, 0.25% deoxycholate, and 0.1% SDS). STAT1 and Jak1/Jak2 molecules were immunoprecipitated by incubation of lysates with polyclonal Abs to 10 µg/ml STAT1 or 10 µg/ml Jak1 and Jak2, respectively. Ab-Ag complexes were captured on protein G-Sepharose beads for 1 h at 4°C. Precipitated proteins were released from the beads by washing in immunoprecipitation buffer (three times) and boiling in SDS sample buffer, and then were separated by SDS-PAGE on a 10% acrylamide-resolving gel. Immunoblot analysis using an anti-phosphorylated-STAT1 Ab (0.5 µg/ml, for STAT1 analysis) or an Ab specific for phophotyrosine residues (1 µg/ml, for Jak1 and Jak2 analysis) was completed essentially as described above. Following analysis of phosphorylated-STAT1, phosphorylated-Jak1, and phosphorylated-Jak2, the blots were stripped and reprobed with Abs directed to STAT1, Jak1, or Jak2, respectively, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VIP and PACAP inhibit IFN-{gamma}-induced IRF-1 expression and its subsequent binding to the iNOS promoter

To investigate whether VIP/PACAP affect IRF-1 nuclear translocation and binding in macrophages stimulated with IFN-{gamma}, we assessed IRF-1 binding to the iNOS promoter by EMSA. Stimulation of Raw 264.7 cells with IFN-{gamma} led to an increase in IRF-1 binding and treatment with VIP or PACAP significantly inhibited this binding (Fig. 1GoA). The specificity of the IRF-1 binding was evident by the complete displacement of the IRF-1/DNA binding complexes in the presence of a 50-fold excess of unlabeled homologous oligonucleotide (IRF-1) but not of nonhomologous oligonucleotide (cAMP response element; Fig. 1GoA). Furthermore, the IRF-1/DNA complexes were supershifted by an anti-IRF-1 Ab in IFN-{gamma}-stimulated macrophages (Fig. 1GoB). Both neuropeptides reduced the IFN-{gamma}-induced presence of IRF-1 protein in the nucleus (Fig. 2GoA). Next, we investigated whether these effects are due to the regulation of IRF-1 expression and synthesis. Northern blots indicated that, although IRF-1 mRNA was not detectable in unstimulated cells, it was strongly induced in the IFN-{gamma}-stimulated Raw 264.7 cells (Fig. 2GoB), and treatment with VIP and PACAP significantly reduced the levels of specific IRF-1 mRNA (Fig. 2GoB). Therefore, these results indicate that VIP and PACAP inhibit IFN-{gamma}-induced IRF-1 expression and its subsequent binding to the promoter of IFN-{gamma}-activated genes in macrophages.



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  		FIGURE 1. VIP and PACAP inhibit IFN-{gamma}-induced IRF-1 binding to the iNOS promoter. A, Nuclear extracts were prepared from 107 Raw 264.7 cells incubated for 2 h with medium alone (unstimulated) or activated with 100 U/ml IFN-{gamma} in the presence or absence of 10-8 M VIP or PACAP. IRF-1 binding was assessed by EMSA using a radiolabeled oligonucleotide containing the murine IRF-1 site of the iNOS promoter. Specificity was assessed by the addition of 50-fold excess of unlabeled homologous (IRF-1), or nonhomologous (NF-{kappa}B) oligonucleotides (Comp). B, Identification of the proteins bound to the IRF-1 site. Nuclear extracts were incubated with polyclonal Abs against IRF-1 or cAMP response element binding protein for 20 min before adding the oligonucleotide probe. The arrow indicates the supershifted IRF-1 band. Similar results were observed in three independent experiments.

 


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  		FIGURE 2. VIP and PACAP inhibit IRF-1 expression in IFN-{gamma}-stimulated macrophages. A, VIP and PACAP decrease the IFN-{gamma}-induced nuclear translocation of IRF-1. Nuclear extracts were prepared from 107 Raw 264.7 cells incubated for 1, 2, or 3 h with medium alone (unstimulated) or with 100 U/ml IFN-{gamma} in the presence or absence of 10-8 M VIP or PACAP. IRF-1 expression was analyzed by Western blot. One representative experiment of three is shown. B, VIP and PACAP inhibit IFN-{gamma}-induced IRF-1 mRNA expression. Raw 264.7 cells (2 x 107 cells) were incubated with 100 U/ml IFN-{gamma} in the presence or absence of 10-8 M VIP or PACAP. Expression of IRF-1 mRNA was analyzed by Northern blot at different times after IFN-{gamma} stimulation. Cells incubated with medium alone were used as basal IRF-1 mRNA-level controls. One representative experiment of three is shown.

 
VIP and PACAP inhibit STAT1 binding to the GAS site of the IRF-1 promoter

Although the IRF-1 promoter contains a complex array of transactivating binding sites, GAS/STAT binding element appears to be essential for maximal IRF-1 gene transcription following IFN-{gamma} stimulation (2). To investigate whether VIP/PACAP affect STAT1 binding to the GAS motif, nuclear extracts from Raw 264.7 macrophages stimulated with IFN-{gamma} in the presence or absence of VIP or PACAP were used in gel shift assays. IFN-{gamma} treatment led to increased binding to an oligonucleotide containing the GAS motif, and treatment with VIP and PACAP inhibited this binding (Fig. 3GoA). The GAS binding was competed by an excess of unlabeled homologous oligonucleotide (GAS) but not by a nonhomologous oligonucleotide (NF-{kappa}B; Fig. 3GoA). Ab supershift experiments indicate that the GAS-binding complexes in IFN-{gamma}-stimulated macrophages contain STAT1 (Fig. 3GoB).



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  		FIGURE 3. VIP and PACAP decrease STAT1 binding to the IRF-1 promoter. A, Raw 264.7 cells (107 cells) were incubated with medium alone (unstimulated) or activated with 100 U/ml IFN-{gamma} in the presence or absence of 10-8 M VIP or PACAP. After 45 min incubation, nuclear extracts were prepared and analyzed for DNA binding activity by EMSA using radiolabeled oligonucleotides containing the GAS sequence motif of the IRF-1 promoter. Specificity was assessed by the addition of 50-fold excess of unlabeled homologous (GAS), or nonhomologous (NF-{kappa}B) oligonucleotides (Comp). B, Identification of the proteins bound to the GAS site. Nuclear extracts were incubated with polyclonal Abs against STAT1 or NF-{kappa}B p65 for 20 min before the addition of the oligonucleotide probe. The arrow indicates the supershifted STAT1 band. Similar results were observed in three independent experiments.

 
The nuclear translocation of STAT1 is preceded by tyrosine phosphorylation and homodimerization of the STAT1{alpha} protein (2, 38, 39). To determine whether VIP and PACAP affect the activation of STAT1, Raw 264.7 cells were stimulated with IFN-{gamma} for different times in the presence or absence of VIP or PACAP. Cell extracts were immunoprecipitated with anti-STAT1 Ab, and the levels of phosphorylated STAT1 were assessed by Western blot using a specific Ab against Tyr701-phosphorylated STAT1{alpha}. IFN-{gamma} stimulated in a time-dependent manner the phosphorylation of STAT1 (Fig. 4Go). Treatment with VIP or PACAP significantly reduced the levels of phosphorylated STAT1 (Fig. 4Go). Therefore, VIP and PACAP inhibit the IFN-{gamma}-induced phosphorylation of STAT1 and its subsequent binding to the GAS site in the IRF-1 gene.



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  		FIGURE 4. VIP and PACAP inhibit the IFN-{gamma}-induced tyrosine phosphorylation of STAT1. Raw 264.7 cells (3 x 107 cells) were incubated with medium alone (unstimulated) or activated with 100 U/ml IFN-{gamma} in the presence or absence of 10-8 M VIP or PACAP. At different times, cell extracts (30 µg protein) were immunoprecipitated (IP) with anti-STAT1 Ab and separated by 10% SDS-PAGE. Proteins were transferred to membranes, and the blots were developed with an Ab specific to phosphorylated STAT1 (upper panel). To determine the levels of STAT1 immunoprecipitated in each case, the membranes were stripped and reprobed with Abs directed to STAT1 (lower panel). Similar results were obtained in three separate experiments.

 
VIP and PACAP reduce IFN-{gamma}-induced Jak1 and Jak2 phosphorylation in Raw 264.7 cells and peritoneal macrophages

STAT1 phosphorylation depends on the activity of the IFN-{gamma}-receptor-associated PTK Jak1/2. Binding of IFN-{gamma} to its receptor results in the activation of Jak1/2 by transphosphorylation (2, 38). To investigate whether VIP and PACAP regulate Jak1 and Jak2 activation, cell extracts of IFN-{gamma}-stimulated Raw 264.7 cells or peritoneal macrophages treated with or without VIP or PACAP were immunoprecipitated with anti-Jak1 or anti-Jak2 Ab, followed by Western blotting with an anti-phosphotyrosine Ab. Both Jak1 and Jak2 were weakly phosphorylated in unstimulated controls; the Jak1/Jak2 phosphorylation level increased within 5 min following IFN-{gamma} stimulation in both Raw 264.7 cells and peritoneal macrophages (Fig. 5Go). Treatment with VIP or PACAP reduced Jak1 and Jak2 phosphorylation at all time points (Fig. 5Go).



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  		FIGURE 5. VIP and PACAP inhibit IFN-{gamma}-induced activation of Jak1 and Jak2 in macrophages. Raw 264.7 cells or peritoneal macrophages from BALB/c mice (3 x 107 cells) were incubated with medium alone (unstimulated) or activated with 100 U/ml IFN-{gamma} in the presence or absence of 10-8 M VIP or PACAP for different time periods. Cell extracts (30 µg protein) were immunoprecipitated (IP) with an anti-Jak1 or anti-Jak2 Ab and resolved by 10% SDS-PAGE. Proteins were transferred to membranes, and immunoblot analysis was conducted using anti-phosphotyrosine (P-Tyr) Abs (upper panel). To determine the levels of immunoprecipitated Jak1 and Jak2, the membranes were stripped and reprobed with Abs against Jak1 or Jak2 (lower panel). Similar results were obtained in three separate experiments.

 
Involvement of VPAC1 and cAMP in the inhibitory effect of VIP on IFN-{gamma}-induced Jak1-STAT1 activation in macrophages

The majority of the effects of VIP and PACAP in macrophages are exerted through the VIP/PACAP-receptor VPAC1, and the subsequent activation of the cAMP/PKA pathway (20, 21, 22, 23, 24, 25, 30, 31, 32, 33). Therefore, we next investigated whether the inhibitory effect of VIP on the Jak/STAT/IRF signaling pathway could be related to occupancy of this specific receptor and to activation of PKA. We investigated the ability of a specific VPAC1-antagonist (40) and of H89, a potent and selective PKA inhibitor, to reverse the effects of VIP. Both the VPAC1 antagonist and the PKA inhibitor reversed the inhibitory effects of VIP on IRF-1 expression (Fig. 6GoA), on the binding of STAT1 to GAS/IRF-1 (Fig. 6GoB), and on STAT1 and Jak1 phosphorylation (Fig. 6GoC). In contrast, PACAP6–38, an antagonist specific for the VIP/PACAP-receptors PAC1 and VPAC2 (41), or calphostin C, a PKC-inhibitor, did not affect the inhibitory effects of VIP (data not shown). These results suggest that the inhibition of the Jak/STAT/IRF signaling pathway by VIP is mediated through VPAC1 and the cAMP/PKA pathway. This is supported by the fact that forskolin (a cAMP-inducing agent) exerts an effect similar to VIP (Fig. 6Go).



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  		FIGURE 6. Involvement of VPAC1 and cAMP signaling in the inhibitory effects of VIP on Jak1-STAT1 activation. Raw 264.7 cells (3 x 107 cells) were activated with 100 U/ml IFN-{gamma} in the absence (lane 1) or presence of 10-8 M VIP (lane 2) or 10-6 M forskolin (lane 5). VPAC1-antagonist (10-6 M, lane 3) or 100 nM H89 (lane 4) were added simultaneously with 10-8 M VIP. A, After 1 h (Northern blot) or 2 h (Western blot), expression of IRF-1 mRNA and nuclear protein was analyzed. Similar results were obtained in three separate experiments. B, After 45 min incubation, nuclear extracts were prepared and analyzed for DNA binding activity by EMSA using radiolabeled oligonucleotides containing the GAS sequence motif of the IRF-1 promoter. Arrow indicates the specific STAT1-GAS complex. One representative experiment of three is shown. C, After 30 min (for STAT1) and 15 min (for Jak1), the phosphorylation of STAT1 and Jak1 was assayed by immunoprecipitation and immunoblotting as described in Figs. 4Go and 5Go, respectively. Similar results were obtained in three independent experiments.

 
VIP/PACAP do not affect SOCS1 and SOCS3 mRNA levels

Because both SOCS1 and 3 inhibit STAT1 activation in response to IFN-{gamma} (42), we investigated the possibility that the effect of VIP/PACAP on Jak/STAT activation is mediated through the induction of SOCS1 and/or SOCS3. Raw 264.7 cells stimulated with LPS or IFN-{gamma} in the presence or absence of VIP/PACAP were analyzed for SOCS1 and SOCS3 expression by Northern blots. As previously reported (43), LPS induces SOCS3 but not SOCS1 expression, whereas IFN-{gamma} induces both (Fig. 7Go). VIP and PACAP do not induce SOCS1/3 in unstimulated macrophages, and do not alter the expression of SOCS1 or SOCS3 in stimulated macrophages at any time point (from 30 min to 20 h; Fig. 7Go).



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  		FIGURE 7. VIP and PACAP do not induce or affect SOCS1 and SOCS3 mRNA levels. Raw 264.7 cells (3 x 107 cells) were incubated with medium alone (unstimulated) or activated with 500 ng/ml LPS or 100 U/ml IFN-{gamma} in the presence or absence of 10-8 M VIP or PACAP for different time periods. Total RNA was extracted and subjected to Northern blot analysis. The probes used were SOCS1, SOCS3, and GAPDH (control for loading). Results are expressed in arbitrary densitometric units normalized per expression of GAPDH in each sample. Upper panel, Results obtained at 1 h. One representative experiment of three is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
VIP and the structurally related neuropeptide PACAP present in the microenvironment of lymphoid organs (16, 44) are potent anti-inflammatory agents that down-regulate the activation of T cells and macrophages (16, 17, 18, 19). Recently, VIP and PACAP were shown to modulate the in vitro and in vivo macrophage secretion of proinflammatory mediators such as TNF-{alpha}, IL-6, IL-12, and NO (20, 21, 22, 24, 25). This might be of clinical relevance, because these cytokines are involved in the detrimental effects of ischemia-reperfusion and septic shock (1, 45, 46, 47, 48). In fact, VIP and PACAP protect mice from lethal endotoxemia, presumably by reducing the levels of endogenous TNF-{alpha} and other macrophage-derived factors (25). VIP/PACAP regulation of proinflammatory cytokines and iNOS occurs at the transcriptional level, and involves several transcriptional factors, such as NF-{kappa}B, cAMP response element binding protein, c-Jun, and IRF-1 (23, 24, 32, 33). In two recent studies, we demonstrated that VIP and PACAP inhibit IL-12 p40 and iNOS mRNA expression in macrophages stimulated with LPS and IFN-{gamma} through two different intracellular pathways; a cAMP-independent pathway that preferentially blocks the LPS-induced NF-{kappa}B nuclear translocation, and a cAMP-dependent pathway that inhibits IFN-{gamma}-induced IRF-1 transactivation (23, 33). NF-{kappa}B activation is prevented through the inhibition of I{kappa}B phosphorylation and degradation, which results in the retention of NF-{kappa}B in the cytoplasm (33). The intracellular mechanisms by which VIP and PACAP inhibit IRF-1 transactivation are not elucidated yet. In the present study we investigated the molecular mechanisms for the suppression of IFN-{gamma}-induced gene expression by VIP/PACAP, with particular emphasis on the role of the Jak1–2/STAT1/IRF-1 signaling pathway. The results demonstrate that VIP and PACAP sequentially inhibit IFN-{gamma}-induced Jak1 and Jak2 activation, and the subsequent activation of STAT1, in terms of phosphorylation, nuclear translocation, and binding to the GAS motif in the IRF-1 promoter. The VIP/PACAP inhibition of IRF-1 synthesis explains in part the inhibitory effect of these neuropeptides on IL-12 and NO production, and suggests a possible role in the regulation of other IFN-{gamma}-stimulated genes. Whether VIP/PACAP affect the response of macrophages to cytokines other than IFN-{gamma} remains to be established. Most cytokine receptors signal through the Jak/STAT family (43) and, therefore, inhibition of Jak1/2 and STAT1 by VIP/PACAP might affect other responses as well. However, although different receptors activate Jak1 and/or Jak2, the ultimate biological response may depend on other factors as well. For example, although both IL-4 and IFN-{gamma} activate Jak1, IL-4 does not affect IRF-1 and actually antagonizes the induction of NO by IFN-{gamma} (50).

The immunological actions of VIP and PACAP are exerted through a family of receptors consisting of VPAC1, VPAC2, and PAC1 (51). Peritoneal macrophages express VPAC1 and PAC1 mRNA constitutively, with VPAC2 mRNA being induced upon LPS stimulation (31, 52, 53). Similar results were obtained for the Raw 264.7 cells (23). Our results demonstrate that VIP exerts its inhibitory effect on the Jak/STAT/IRF signaling pathway through VPAC1. This finding is in agreement with previous reports that VPAC1 mediates VIP inhibition of LPS/IFN-{gamma}-induced IL-12 and iNOS expression (23, 31, 33).

VPAC1 is coupled primarily to the adenylate cyclase system (51), and Jak1-STAT1 activation is indeed inhibited by agents that increase intracellular cAMP (54, 55, 56, 57). In the present study, forskolin, a cAMP-inducing agent, inhibited Jak1 activation, STAT1 phosphorylation and binding to the GAS motif, and IRF-1 synthesis. In addition, H89, a potent and selective PKA inhibitor, reversed the inhibitory effect of VIP on Jak1/STAT1 and IRF-1 synthesis, supporting the involvement of the cAMP/PKA pathway. Similar observations were made for the inhibitory effect of NO and IL-12 production by VIP in LPS/IFN-{gamma}-stimulated macrophages (23, 33). The link between the cAMP pathway and the Jak1/2 activation has not been elucidated. One of the possibilities is the induction of SOCS1 and/or SOCS3 by VIP/PACAP. The members of the SOCS family act as inhibitors of cytokine signaling (58, 59, 60), and SOCS1 and SOCS3 inhibit STAT1 activation by IFN-{gamma} (42). However, VIP and PACAP did not induce the expression of SOCS1 and SOCS3 in unstimulated macrophages, and did not alter SOCS1 and/or SOCS3 expression in cells treated with either LPS or IFN-{gamma}.

Several neurohormones activate specific members of the Jak/STAT family. Prolactin activates Jak2, STAT1 and STAT5 (61, 62); the growth hormone activates Jak1 and Jak2, followed by STAT1, STAT3, and STAT5 (63); and {alpha}-melanocyte-stimulating hormone activates Jak2 and STAT1 (64). However, no neuropeptides or hormones have been previously reported to inhibit the Jak/STAT pathway. To our knowledge, VIP and PACAP are the first example of neuropeptides that negatively regulate the expression of IRF-1 through the inhibition of Jak1/STAT1 activation.

IFN-{gamma} is one of the most important macrophage stimulators in vivo, and STAT1 and IRF-1 play central roles in the expression of several genes essential for both innate and specific immunity. Therefore, the regulation of Jak/STAT1/IRF activation by VIP and PACAP could represent an important regulatory element in the complex machinery by which these endogenous neuropeptides regulate the inflammatory response.


   Footnotes
 
1 This work was supported by Public Health Service Grant AI 041786-01 (to D.G.), Busch Biomedical Award 98-00 (to D.G.), Grant PM98-0081 (to M.D.), and a postdoctoral fellowship from the Spanish Department of Education and Science (M.D.). Back

2 Address correspondence and reprint requests to Dr. Doina Ganea, Rutgers University, Department of Biological Sciences, 101 Warren Street, Newark, NJ 07102. Back

3 Abbreviations used in this paper: Jak, Janus kinase; GAS, IFN-{gamma}-activated site; IRF-1, IFN regulatory factor-1; iNOS, inducible NO synthase; PAC1, PACAP-preferring receptor; PACAP, pituitary adenylate cyclase-activating polypeptide; SOCS, suppressor of cytokine signaling; VIP, vasoactive intestinal peptide, VPAC, VIP/PACAP receptor; PKA, protein kinase A. Back

Received for publication February 14, 2000. Accepted for publication June 29, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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