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• W2081736295 abstract "Interferon (IFN)-γ is one of the most important microglia stimulators in vivo participating in inflammation and Th1 activation/differentiation. IFN-γ-mediated signaling involves the activation of the Jak/STAT1 pathway. The neuropeptides vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptide (PACAP) are two potent microglia-deactivating factors that inhibit the production of proinflammatory mediators in vitro and in vivo. The present study investigated the molecular mechanisms involved in the VIP/PACAP regulation of several IFN-γ-induced microglia-derived factors, including IFN-γ-inducible protein-10 (IP-10), inducible nitric-oxide synthase (iNOS), and CD40. The results indicate that VIP/PACAP inhibit Jak1–2 and STAT1 phosphorylation, and the binding of activated STAT1 to the IFN-γ activated site motif in the IFN regulatory factor-1 and CD40 promoter and to the IFN-stimulated response element motif of the IP-10 promoter. Through its effect in the IFN-γ-induced Jak/STAT1 pathway, VIP and PACAP are able to control the gene expression of IP-10, CD40, and iNOS, three microglia-derived mediators that play an essential role in several pathologies, i.e. inflammation and autoimmune disorders. The effects of VIP/PACAP are mediated through the specific receptor VPAC1 and the cAMP/protein kinase A transduction pathway. Because IFN-γ is a major stimulator of innate and adaptive immune responses in vivo, the down-regulation of IFN-γ-induced gene expression by VIP and PACAP could represent a significant element in the regulation of the inflammatory response in the central nervous system by endogenous neuropeptides. Interferon (IFN)-γ is one of the most important microglia stimulators in vivo participating in inflammation and Th1 activation/differentiation. IFN-γ-mediated signaling involves the activation of the Jak/STAT1 pathway. The neuropeptides vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptide (PACAP) are two potent microglia-deactivating factors that inhibit the production of proinflammatory mediators in vitro and in vivo. The present study investigated the molecular mechanisms involved in the VIP/PACAP regulation of several IFN-γ-induced microglia-derived factors, including IFN-γ-inducible protein-10 (IP-10), inducible nitric-oxide synthase (iNOS), and CD40. The results indicate that VIP/PACAP inhibit Jak1–2 and STAT1 phosphorylation, and the binding of activated STAT1 to the IFN-γ activated site motif in the IFN regulatory factor-1 and CD40 promoter and to the IFN-stimulated response element motif of the IP-10 promoter. Through its effect in the IFN-γ-induced Jak/STAT1 pathway, VIP and PACAP are able to control the gene expression of IP-10, CD40, and iNOS, three microglia-derived mediators that play an essential role in several pathologies, i.e. inflammation and autoimmune disorders. The effects of VIP/PACAP are mediated through the specific receptor VPAC1 and the cAMP/protein kinase A transduction pathway. Because IFN-γ is a major stimulator of innate and adaptive immune responses in vivo, the down-regulation of IFN-γ-induced gene expression by VIP and PACAP could represent a significant element in the regulation of the inflammatory response in the central nervous system by endogenous neuropeptides. Under normal conditions, brain microglia are involved in immune surveillance and host defense against infectious agents (1Dixon D.W. Mattiace L.A. Kure K. Hutchins K. Lyman X. Brosnan C.F. Lab. Invest. 1991; 64: 135-156PubMed Google Scholar). However, in response to brain injury, infection, or inflammation, microglia readily become activated, in a way similar to peripheral tissue macrophages, a process that includes differentiation and probably invasion and proliferation. Activation of microglia is a histopathological hallmark of several neurodegenerative diseases, including Alzheimer and Parkinson's diseases, multiple sclerosis, and the AIDS dementia complex (1Dixon D.W. Mattiace L.A. Kure K. Hutchins K. Lyman X. Brosnan C.F. Lab. Invest. 1991; 64: 135-156PubMed Google Scholar, 2Streit W.J. Graeber M.B. Kreutzberg G.W. Glia. 1988; 1: 301-307Crossref PubMed Scopus (856) Google Scholar, 3Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (894) Google Scholar). Pathological microglial activation is believed to contribute to progressive damage in neurodegenerative diseases through the release of proinflammatory and/or cytotoxic factors, including TNF-α, 1The abbreviations used are: TNF-α, tumor necrosis factor α; GAS, interferon γ-activated site; IFN, interferon; IRF-1, interferon regulatory factor 1; NO, nitric oxide; iNOS, inducible nitric-oxide synthase; IP-10, interferon-induced protein-10; ISRE, interferon-stimulated response element; PAC1, pituitary adenylate cyclase activating polypeptide-preferring receptor; PACAP, pituitary adenylate cyclase activating polypeptide; STAT1, signal transducer and activator of transcription 1; VIP, vasoactive intestinal peptide; VPAC1 and VPAC2, vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide receptors 1 and 2; IL, interleukin; Ab, antibody; PBS, phosphate-buffered saline; SOCS, suppressors of cytokine signaling; TBS-T, Tris-buffered saline with Tween 20; SBE, signal transducer and activator of transcription binding element; LPS, lipopolysaccharide; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PKA, cAMP-dependent protein kinase; EMSA, electrophoretic mobility shift assay; ELISA, enzyme-linked immunosorbent assay; EAE, experimental autoimmune encephalomyelitis. IL-1β, IL-6, IL-12, and nitric oxide. Hence, it is important to unravel mechanisms regulating microglia activation of inflamed brain parenchyma to provide insights into efficient therapeutic intervention. IFN-γ constitutes one of the most potent microglia-activating factors. Binding of IFN-γ to its receptor induces the assembly of an active receptor complex and consequent transphosphorylation of the receptor-associated Janus tyrosine kinases Jak1 and Jak2 (4Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5028) Google Scholar, 5Greenlund A.C. Morales M.O. Viviano B.L. Yan H. Krolewski J. Schreiber R.D. Immunity. 1995; 2: 677-687Abstract Full Text PDF PubMed Scopus (250) Google Scholar). The activation of these kinases induces phosphorylation of the cytoplasmic tail of the receptor itself, which lacks intrinsic kinase activity. The cytosolic protein signal transducer and activator of transcription (STAT1) is then recruited to the activated IFN-γ-receptor complex and phosphorylated (4Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5028) Google Scholar). Upon phosphorylation, STAT1 forms homodimers and translocate to the nucleus, where they bind to the IFN-γ-activated site (GAS), also termed STAT binding element (SBE), found in the promoter of many IFN-γ-induced genes including the IFN regulatory factor-1 (IRF-1) and ICAM-1 genes (6Sims S.H. Cha Y. Romine M.F. Gao P. Gottlieb K. Deisseroth A.B. Mol. Cell. Biol. 1993; 13: 690-702Crossref PubMed Scopus (249) Google Scholar, 7Pine R. Canova A. Schindler C. EMBO J. 1994; 13: 158-167Crossref PubMed Scopus (340) Google Scholar, 8Caldenhoven E. Coffer P. Yuan J. van de Stolpe A. Horn F. Kruijer W. Van Der Saag P.T. J. Biol. Chem. 1994; 269: 21146-21154Abstract Full Text PDF PubMed Google Scholar, 9Look D.C. Pelletier M.R. Holtzman M.J. J. Biol. Chem. 1994; 269: 8952-8958Abstract Full Text PDF PubMed Google Scholar). Many of the regulatory effects of IFN-γ in microglia appear to be mediated by IRF-1 and/or STAT1, which transactivate multiple effector genes including IFN-γ-inducible protein-10 (IP-10), CD40, IL-12, and the inducible nitric-oxide synthase (iNOS) (10Ma X. Neurath M. Gri G. Trinchieri G. J. Biol. Chem. 1997; 272: 10389-10395Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 11Lowenstein C.J. Alley E.W. Raval P. Snowman A.M. Synder S.H. Russel S.W. Murphy W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9730-9734Crossref PubMed Scopus (1008) Google Scholar, 12Xie Q.W. Wishnan R. Nathan C. J. Exp. Med. 1993; 177: 1779-1784Crossref PubMed Scopus (1030) Google Scholar, 13Chartrain N.S. Geller D.A. Koty P.P. Sitrin N.F. Nussler A.K. Hoffman E.P. Billiar T.R. Hutchinson N.I. Mudgett J.S. J. Biol. Chem. 1994; 269: 6765-6772Abstract Full Text PDF PubMed Google Scholar, 14Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D. Le J. Koh S.I. Kimura T. Green S.J. Mak T.W. Taniguchi T. Vilcek J. Science. 1994; 264: 1612-1615Crossref Scopus (787) Google Scholar, 15Martin E. Nathan C. Xie Q.W. J. Exp. Med. 1994; 180: 977-984Crossref PubMed Scopus (456) Google Scholar, 16Ohmori Y. Hamilton T.A. J. Biol. Chem. 1993; 268: 6677-6688Abstract Full Text PDF PubMed Google Scholar, 17Ohmori Y. Hamilton T.A. J. Immunol. 1995; 154: 5235-5244PubMed Google Scholar, 18Vanguri P. Farber J.M. J. Immunol. 1994; 152: 1411-1418PubMed Google Scholar, 19Nguyen V.T. Benveniste E.N. J. Biol. Chem. 2002; 277: 13796-13803Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 20Nguyen V.T. Benveniste E.N. J. Biol. Chem. 2000; 275: 23674-23684Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Vasoactive intestinal peptide (VIP) and the structurally related peptide, the pituitary adenylate cyclase activating polypeptide (PACAP), are two neuropeptides that elicit a broad spectrum of biological functions, including actions on natural and acquired immunity (reviewed in Refs. 21Pozo D. Delgado M. Martinez C. Guerrero J.M. Leceta J. Gomariz R.P. Calvo J.R. Immunol. Today. 2000; 21: 7-11Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 22Delgado M. Abad C. Martinez C. Juarranz M.G. Arranz A. Gomariz R.P. Leceta J. J. Mol. Med. 2002; 80: 16-24Crossref PubMed Scopus (143) Google Scholar, 23Gomariz R.P. Martinez C. Abad C. Leceta J. Delgado M. Curr. Pharm. Design. 2001; 7: 89-111Crossref PubMed Scopus (156) Google Scholar, 24Ganea D. Delgado M. Crit. Rev. Oral Biol. Med. 2002; 13: 229-237Crossref PubMed Scopus (131) Google Scholar, 25Goetzl E.J. Pankhaniya R.R. Gaufo G.O. Mu Y. Xia M. Sreedharan S.P. Ann. N. Y. Acad. Sci. 1998; 840: 540-550Crossref PubMed Scopus (40) Google Scholar). Although VIP and PACAP affect a variety of immune functions, their primary immunomodulatory function is anti-inflammatory in nature. VIP has been shown to inhibit cytokine production and proliferation in T cells, and several macrophage functions, including phagocytosis, respiratory burst, and chemotaxis (23Gomariz R.P. Martinez C. Abad C. Leceta J. Delgado M. Curr. Pharm. Design. 2001; 7: 89-111Crossref PubMed Scopus (156) Google Scholar), as well as LPS-induced IL-6, TNF-α, IL-12, NO, and chemokine production (21Pozo D. Delgado M. Martinez C. Guerrero J.M. Leceta J. Gomariz R.P. Calvo J.R. Immunol. Today. 2000; 21: 7-11Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 22Delgado M. Abad C. Martinez C. Juarranz M.G. Arranz A. Gomariz R.P. Leceta J. J. Mol. Med. 2002; 80: 16-24Crossref PubMed Scopus (143) Google Scholar, 23Gomariz R.P. Martinez C. Abad C. Leceta J. Delgado M. Curr. Pharm. Design. 2001; 7: 89-111Crossref PubMed Scopus (156) Google Scholar, 24Ganea D. Delgado M. Crit. Rev. Oral Biol. Med. 2002; 13: 229-237Crossref PubMed Scopus (131) Google Scholar, 25Goetzl E.J. Pankhaniya R.R. Gaufo G.O. Mu Y. Xia M. Sreedharan S.P. Ann. N. Y. Acad. Sci. 1998; 840: 540-550Crossref PubMed Scopus (40) Google Scholar). Similarly, we have recently demonstrated that VIP and PACAP act as potent microglia-deactivating factors by inhibiting the production of endotoxin-induced proinflammatory mediators in vitro (26Delgado M. Jonakait G.M. Ganea D. Glia. 2002; 39: 148-161Crossref PubMed Scopus (115) Google Scholar, 27Delgado M. Leceta J. Ganea D. J. Leukocyte Biol. 2003; 73: 155-164Crossref PubMed Scopus (120) Google Scholar). The inhibition of proinflammatory mediators is responsible, at least partially, for the protective effect of VIP/PACAP in vivo in murine models for septic shock, inflammation-induced neurodegeneration, brain trauma, and Parkinson's disease (28Delgado M. Martinez C. Pozo D. Calvo J.R. Leceta J. Ganea D. Gomariz R.P. J. Immunol. 1999; 162: 1200-1205PubMed Google Scholar, 29Delgado M. Ganea D. FASEB J. 2003; 17: 944-946Crossref PubMed Scopus (152) Google Scholar, 30Delgado, M., and Ganea, D. (2003) FASEB J., in pressGoogle Scholar). To further understand the molecular mechanisms through which VIP and PACAP attenuate the inflammatory responses in the central nervous system, we examined the effects of VIP/PACAP on IFN-γ-induced Jak-STAT1 activation and IRF-1 synthesis in murine microglial cells. Our results indicate that VIP/PACAP inhibit Jak1–2/STAT1 phosphorylation, binding of STAT1 to the GAS motif in the IRF-1 promoter, and subsequently, IRF-1 transcription and synthesis. These effects are correlated with an inhibitory effect of VIP/PACAP on IFN-γ-induced CD40, IP-10, and iNOS expression, mediated through the VIP receptor VPAC1 and the cAMP/PKA transduction pathway. Reagents—Synthetic VIP, helodermin, VIP10–28, and PACAP38 were purchased from Novabiochem (Laufelfingen, Switzerland). The VPAC1 antagonist [Ac-His1,d-Phe2,Lys15,Arg16,Leu27[VIP-(3–7)-GRF-(8–27), the VPAC1 agonist [Lys15,Arg16,Leu27]VIP-(1–7)-GRF-(8–27), and the VPAC2 agonist Ro-25-1553 Ac-[Glu8,Lys12,Nle17,Ala19,Asp25,Leu-26,Lys27,Lys28,Gly29,Gly30,Thr31]VIP cyclo (21Pozo D. Delgado M. Martinez C. Guerrero J.M. Leceta J. Gomariz R.P. Calvo J.R. Immunol. Today. 2000; 21: 7-11Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 22Delgado M. Abad C. Martinez C. Juarranz M.G. Arranz A. Gomariz R.P. Leceta J. J. Mol. Med. 2002; 80: 16-24Crossref PubMed Scopus (143) Google Scholar, 23Gomariz R.P. Martinez C. Abad C. Leceta J. Delgado M. Curr. Pharm. Design. 2001; 7: 89-111Crossref PubMed Scopus (156) Google Scholar, 24Ganea D. Delgado M. Crit. Rev. Oral Biol. Med. 2002; 13: 229-237Crossref PubMed Scopus (131) Google Scholar, 25Goetzl E.J. Pankhaniya R.R. Gaufo G.O. Mu Y. Xia M. Sreedharan S.P. Ann. N. Y. Acad. Sci. 1998; 840: 540-550Crossref PubMed Scopus (40) Google Scholar) were 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-γ and antibodies against CD40 and IP-10 were purchased from Pharmingen. Forskolin, protease inhibitors, flavin-adenine dinucleotide, sodium pyruvate, tetrahydrobioptein, l-lactic dehydrogenase, NADPH, l-arginine, sulfanilamide, N-[naphthyl]ethylenediamine dihydrochloride, PMSF, EDTA, glycine, protein G-Sepharose, glycerol, EGTA, and DTT were purchased from Sigma, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) from ICN Pharmaceuticals Inc (Costa Mesa, CA). Antibodies against IRF-1, phospho-Tyr (PY20), phosphorylated STAT1 (Tyr701), Jak1, Jak2, STAT1α p91, STAT2, and NFκB p65 were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Monoclonal antibody against mouse iNOS was purchased from Transduction Laboratories (Lexington, KY). Antibody against phosphorylated STAT1 (Ser727) was obtained from Upstate Biotechnology (Lake Placid, NY). Cell Cultures—Microglial cell cultures were prepared as previously described (31Chao C.C. Molitor T.W. Shuxian H. J. Immunol. 1993; 151: 1473-1481PubMed Google Scholar). Briefly, cerebral cortical cells from 1-day-old BALB/c mice were dissociated after a 30-min trypsinization (0.25%) and were plated in 75-cm2 Falcon culture flasks in Dulbecco's modified Eagle's medium high glucose formula (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen), containing 10 mm HEPES buffer, 1 mm pyruvate, 0.1 m nonessential amino acids, 2 mm glutamine, 50 mm 2-mercaptoethanol, 100 units/ml penicillin, and 10 μg/ml streptomycin (complete medium). The medium was replenished 1 and 4 days after plating, and on day 8 of culture, plates were shaken for 20 min at a speed of 200 rpm in an orbital shaker to remove oligodendrocytes. On day 12 of culture, plates were shaken again for 2 h at a speed of 180–200 rpm. Harvested cells were filtered through a 20-μm nylon mesh, plated in a 60-mm Petri dish, and incubated for 15 min at 37 °C. After extensive washing with culture medium, adherent cells (microglia) were collected with a rubber policeman and centrifuged (1000 rpm, 10 min). Purified microglial cell cultures comprised a cell population in which >98% stained positively with MAC-1 antibodies (Roche Molecular Biochemicals) and <2% stained positively with antibodies specific to the astrocyte marker glial fibrillary acid protein (Sigma). Microglia monolayers were incubated with complete medium and stimulated with 50 units/ml IFN-γ in the presence or absence of VIP or PACAP38 (from 10–12 to 10–6m) 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 IP-10 determination. RNA Extraction and Northern Blot Analysis—Northern blot analysis was performed according to standard methods. Murine primary microglia cells were cultured at a concentration of 2 × 106 cells/ml in 100-mm tissue culture dishes and stimulated with IFN-γ (50 units/ml) in the presence or absence of VIP (10–8m) or PACAP (10–8m) for different time periods at 37 °C. Cells were collected, and total RNA was extracted by the acid guanidinium-phenol-chloroform method, electrophoresed on 1.2% agarose-formaldehyde gels, transferred to Nytran membranes (Schleicher & Schuell), and cross-linked to the nylon membrane using UV light. The probes for murine IRF-1, GAPDH, and IP-10 were generated by reverse transcription-PCR as previously described (32Miyamoto M. Fujita T. Kimura Y. Maruyama M. Harada H. Sudo Y. Miyata T. Taniguchi T. Cell. 1988; 54: 903-913Abstract Full Text PDF PubMed Scopus (795) Google Scholar, 33Kuroda E. Sugiura T. Okada K. Zeki K. Yamashita U. J. Immunol. 2001; 166: 1650-1658Crossref PubMed Scopus (67) Google Scholar). Oligonucleotides were end-labeled with [γ-32P]ATP (3000 Ci/mmol, Amersham Biosciences) 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 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 Inc. (Boulder, CO). The membranes were exposed to x-ray films (Eastman Kodak Co.). CD40 mRNA expression was determined by RNase protection assay as described previously (34Nguyen V.T. Walker W.S. Benveniste E.N. Eur. J. Immunol. 1998; 28: 2537-2548Crossref PubMed Scopus (84) Google Scholar). Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts were prepared by the mini-extraction procedure of Schreiber et al. (35Schreiber E. Metthias P. Muller M.M. Shaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3917) Google Scholar) with slight modifications. Briefly, microglia cells were cultured at a density of 107 cells in 6-well plates, stimulated as described above, washed twice with ice-cold PBS plus 0.1% bovine serum albumin, and harvested from the dishes. Incubation times for the different factors assayed were optimized empirically. 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 × 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 IFN stimulus response element (ISRE) (nucleotides –234 to –206) motif of the IP-10 promoter (16Ohmori Y. Hamilton T.A. J. Biol. Chem. 1993; 268: 6677-6688Abstract Full Text PDF PubMed Google Scholar, 17Ohmori Y. Hamilton T.A. J. Immunol. 1995; 154: 5235-5244PubMed Google Scholar, 36Nazar A.S.M.I. Cheng G. Shin H.S. Brothers P.N. Dhib-Jalbut S. Shin M.L. Vanguri P. J. Neuroimmunol. 1997; 77: 116-127Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), to the IRF-1 (nucleotides –933 to –906) motif of the iNOS promoter (11Lowenstein C.J. Alley E.W. Raval P. Snowman A.M. Synder S.H. Russel S.W. Murphy W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9730-9734Crossref PubMed Scopus (1008) Google Scholar, 12Xie Q.W. Wishnan R. Nathan C. J. Exp. Med. 1993; 177: 1779-1784Crossref PubMed Scopus (1030) Google Scholar), to the mGAS (nucleotides –491 to –467) site of CD40 promoter (19Nguyen V.T. Benveniste E.N. J. Biol. Chem. 2002; 277: 13796-13803Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 20Nguyen V.T. Benveniste E.N. J. Biol. Chem. 2000; 275: 23674-23684Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), and to the GAS site of IRF-1 promoter were synthesized (nucleotides –131 to –105) (6Sims S.H. Cha Y. Romine M.F. Gao P. Gottlieb K. Deisseroth A.B. Mol. Cell. Biol. 1993; 13: 690-702Crossref PubMed Scopus (249) Google Scholar): 5′-CTCACGCTTTGGAAAGTGAAACCTACCTC-3′ (ISRE for IP-10), 5′-CACTGTCAATATTTCACTTTCATAATG-3′ (IRF-1 for iNOS), 5′-GGAAACTCTTCCTTGAAACGCCTCC-3′ (GAS for CD40), and 5′-GCCTGATTTCCCCGAAATGACGGC-3′ (GAS for IRF-1). The oligonucleotides were annealed after incubation for 5 min at 85 °C in 10 mm Tris-HCl, pH 8.0, 5 mm NaCl, 10 mm MgCl2, and 1 mm DTT. Aliquots of 50 ng of the double-stranded oligonucleotides were end-labeled with [γ-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 binding reaction mixtures (15 μl) were set up containing: 0.5–1 ng of DNA probe, 5 μg of nuclear extract, 2 μg of poly(dI-dC)·poly(dI-dC), and binding buffer (50 mm NaCl, 0.2 mm EDTA, 0.5 mm DTT, 5% glycerol, and 10 mm Tris-HCl, pH 7.5). The mixtures were incubated on ice for 15 min before adding the probe, followed by another 20 min at room temperature. Samples were loaded onto 4% nondenaturing polyacrylamide gels and electrophoresed in TGE buffer (50 mm Tris-HCl, pH 7.5, 0.38 m glycine, and 2 mm EDTA) at 100 V, followed by transfer to Whatman paper, drying under vacuum at 80 °C, and autoradiography. In competition and antibody supershift experiments, the nuclear extracts were incubated for 15 min at room temperature with the specific antibody (1 μg) or competing cold oligonucleotide (50-fold excess) before the addition of the labeled probe. Western Blot Analysis of iNOS—For detection of iNOS by Western blotting, microglia cells (107 cells) were lysed in cold 40 mm Tris-HCl buffer, pH 8.0, containing 0.1 mm PMSF, 5 μg/ml aprotinin, 50 μg/ml leupeptin, 1 μg/ml chymostatin, and 5 μg/ml pepstatin, and lysed by sonication. Thirty micrograms of protein extracts was separated by 7.5% SDS-PAGE gels under reducing conditions. 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 TBS-T buffer (10 mm Tris, pH 8.0, 150 mm NaCl, 0.05% Tween 20) containing 5% milk powder for1hat room temperature, then incubated for an additional 6 h at 4 °C with a monoclonal antibody against iNOS (1:1000) in 5% nonfat milk/TBS-T solution. After washing three times in TBS-T, membranes were incubated at room temperature for 1 h with peroxidase-conjugated goat anti-mouse IgG at 1:2000 dilution. After washing three times in TBS-T for 5 min each, and once in Tris-buffered saline for 5 min, the membrane was drained briefly and subjected to the enhanced chemiluminescence detection system (ECL, Amersham Biosciences). 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 (38Lucas D.M. Lokuta M.A. McDowell M.A. Doan J.E.S. Paulnock D.M. J. Immunol. 1998; 160: 4337-4342PubMed Google Scholar). Cells (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 β-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 STAT1 (10 μg/ml) or Jak1 and Jak2 (10 μg/ml), respectively. Ab-antigen 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 separated by SDS-PAGE on a 10% acrylamide resolving gel. Immunoblot analysis using an anti-phosphorylated STAT1 Abs (0.5 μg/ml, for Tyr701 and Ser727 STAT1 analysis) or an Ab specific for phosphotyrosine residues (1 μg/ml, for Jak1 and Jak2 analysis) was done 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. IP-10 ELISA—The amounts of IP-10 present in culture supernatants were determined using a specific sandwich ELISA. Briefly, a capture monoclonal anti-murine IP-10 antibody (clone A 102–6, 2.5 μg/ml) was used for coating, and a biotinylated rabbit anti-mouse IP-10 polyclonal antibody (4 μg/ml) was used for detection. The detection limit for the assay is 15 pg/ml IP-10. Determination of NO Synthase Activity—The NO synthase activity was measured as described (39Delgado M. Munoz-Elias E.J. Gomariz R.P. Ganea D. J. Immunol. 1999; 162: 4685-4696PubMed Google Scholar). Briefly, primary microglia cells were cultured at a density of 107 cells in 6-well plates, stimulated as described above, washed twice with ice-cold PBS plus 0.1% bovine serum albumin. Cells were lysed by three cycles of freeze/thaw in 40 mm Tris-HCl, pH 8.0, 0.1 mm EGTA, 0.1 mm EDTA, 12 mm 2-mercaptoethanol, and protease inhibitors (0.1 mm PMSF, 5 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml chymostatin, and 5 μg/ml pepstatin), and centrifuged at 10,000 × g for 60 min. For the NO synthase activity assay, microglia lysates were incubated with 2 mm l-arginine, 2 mm NADPH, 4 μm flavin-adenine dinucleotide, 4 μm tetrahydrobiopterin, 2 mm DTT, and1mm EGTA for2hat37 °C.The reaction was terminated by adding 15 units/ml l-lactic dehydrogenase and 83 mm sodium pyruvate and incubating for an additional 15 min. The amount of NO formed in the reaction mixture was estimated from the accumulation of the stable NO metabolite nitrite by the Griess assay. Equal volumes of culture supernatants and Griess reagents (1% sulfanilamide, 0.1% N-[naphthyl]ethylenediamine dihydrochloride in 2.5% H3PO4) were mixed, and the absorbance was measured at 550 nm. The amount of nitrite was calculated from a NaNO2 standard curve. Determination of CD40 Protein Expression by Immunofluorescence Flow Cytometry—CD40 expression was determined by flow cytometry as previously described (20Nguyen V.T. Benveniste E.N. J. Biol. Chem. 2000; 275: 23674-23684Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Briefly, primary microglia were plated at 2 × 105 cells/well into 12-well plates (Costar) and stimulated as described above. The cells were scraped in ice-cold RPMI complete medium and washed twice with PBS containing 0.1% sodium azide plus 2% heat-inactivated fetal calf serum (wash buffer). Cells were incubated with 10 μg/ml rat IgG2a-k anti-mouse CD40 Ab (clone 3/23) at 4 °C for 45 min, then washed, and incubated with 10 μg/ml biotinylated anti-rat IgG2a, for 30 min at 4 °C. After washing, the cells were incubated with 10 μg/ml phycoerythrin-conjugated streptavidin for 30 min at 4 °C. Isotype-matched Abs were used as controls, and IgG block (Sigma) was used to block the nonspecific binding to Fc receptors. After extensive washing, the cells were fixed in 1% paraformaldehyde. Stained microglia cells, gated according to forward and side scatter characteristics, were analyzed on a FACScan flow cytometer (Becton Dickinson). Samples in which isotype-matched antibody was used instead of specific antibody were used as negative controls to determine the proper region or window setting. Fluorescence data were expressed as mean fluorescence intensity and as percentage (%) of positive cells after subtraction of background isotype-matched values. VIP and the structurally related neuropeptide PACAP have been lately identified as two potent anti-inflammatory agents, which down-regulate the activation of T cells and macrophages (reviewed in Refs. 21Pozo D. Delgado M. Martinez C. Guerrero J.M. Leceta J. Gomariz R.P. Calvo J.R. Immunol. Today. 2000; 21: 7-11Abstract Full Text Full Text PDF PubMed Sco" @default.
• W2081736295 created "2016-06-24" @default.
• W2081736295 creator A5091295224 @default.
• W2081736295 date "2003-07-01" @default.
• W2081736295 modified "2023-10-05" @default.
• W2081736295 title "Inhibition of Interferon (IFN) γ-induced Jak-STAT1 Activation in Microglia by Vasoactive Intestinal Peptide" @default.
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• W2081736295 doi "https://doi.org/10.1074/jbc.m303199200" @default.
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• W2081736295 hasPublicationYear "2003" @default.
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