Constitutive Activation of the Signal Transducer and Activator of Transcription Pathway in Celiac Disease Lesions
2003; Elsevier BV; Volume: 162; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64319-2
ISSN1525-2191
AutoresGiuseppe Mazzarella, Thomas T. MacDonald, V.M. Salvati, Peter Mulligan, Luigi Pasquale, Rosita Stefanile, Paolo Lionetti, Salvatore Auricchio, Francesco Pallone, Riccardo Troncone, Giovanni Monteleone,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoThe biological effects of interferon (IFN)-γ rely mainly on the activity of the transcription factor signal transducer and activator of transcription (STAT) 1 and the intracellular levels of suppressor of cytokine signaling (SOCS)-1, a negative regulator that controls the amplitude and duration of STAT-1 activation. IFN-γ is a key mediator of the immunopathology in celiac disease (CD, gluten-sensitive enteropathy). Thus we have investigated STAT-1 signaling and SOCS-1 expression in this condition. As expected, high local concentrations of IFN-γ were invariably seen in duodenal biopsies from CD patients in comparison to controls. On the basis of immunohistochemistry, STAT-1 phosphorylation, nuclear localization, and DNA-binding activity, STAT-1 activation was consistently more pronounced in CD compared with controls. Despite samples from CD patients containing abundant SOCS-1 mRNA, SOCS-1 protein was expressed at the same level in CD patients and controls. In explant cultures of CD biopsies, gliadin induced the activation of STAT-1 but not SOCS-1. Furthermore, inhibition of STAT-1 prevented the gliadin-mediated induction of ICAM-1 and B7-2. These data suggest that persistent STAT-1 activation can contribute to maintaining and expanding the local inflammatory response in CD. The biological effects of interferon (IFN)-γ rely mainly on the activity of the transcription factor signal transducer and activator of transcription (STAT) 1 and the intracellular levels of suppressor of cytokine signaling (SOCS)-1, a negative regulator that controls the amplitude and duration of STAT-1 activation. IFN-γ is a key mediator of the immunopathology in celiac disease (CD, gluten-sensitive enteropathy). Thus we have investigated STAT-1 signaling and SOCS-1 expression in this condition. As expected, high local concentrations of IFN-γ were invariably seen in duodenal biopsies from CD patients in comparison to controls. On the basis of immunohistochemistry, STAT-1 phosphorylation, nuclear localization, and DNA-binding activity, STAT-1 activation was consistently more pronounced in CD compared with controls. Despite samples from CD patients containing abundant SOCS-1 mRNA, SOCS-1 protein was expressed at the same level in CD patients and controls. In explant cultures of CD biopsies, gliadin induced the activation of STAT-1 but not SOCS-1. Furthermore, inhibition of STAT-1 prevented the gliadin-mediated induction of ICAM-1 and B7-2. These data suggest that persistent STAT-1 activation can contribute to maintaining and expanding the local inflammatory response in CD. The major signal transduction pathway initiated by interferon (IFN)-γ has been elucidated by a series of elegant biochemical and genetic studies. Binding of IFN-γ to its receptor induces dimerization of the receptor chains, bringing together JAK kinases 1 and 2 that are activated by transphosphorylation. Activated JAK-1 and -2 then phosphorylate tyrosine residues within the cytoplasmic domains of the receptor subunits, which act as a docking site for a latent cytoplasmic protein, termed signal transducer and activator of transcription 1 (STAT-1). Phosphorylation of a C-terminal tyrosine (Y701) in STAT-1 facilitates interaction with the SH2 domain of a second STAT-1 molecule, mediating dimerization. STAT-1 can also be phosphorylated on a single serine residue (Ser-727) at the carboxy end of the molecule.1Janeway CA Bottolmy K Signals and signs for lymphocyte responses.Cell. 1994; 76: 275-285Abstract Full Text PDF PubMed Scopus (766) Google Scholar, 2Horvath CM Darnell Jr, JE The state of the STATs: recent developments in the study of signal transduction to the nucleus.Curr Opin Cell Biol. 1997; 9: 233-239Crossref PubMed Scopus (173) Google ScholarSTAT-1 dimers subsequently migrate to the nucleus, where they bind to the γ-activated sequence (GAS) element contained within the promoters of IFN-γ-inducible immune-inflammatory genes (eg, IRF-1, INOS, CD86, MHC class II antigens, ICAM-1).1Janeway CA Bottolmy K Signals and signs for lymphocyte responses.Cell. 1994; 76: 275-285Abstract Full Text PDF PubMed Scopus (766) Google Scholar, 2Horvath CM Darnell Jr, JE The state of the STATs: recent developments in the study of signal transduction to the nucleus.Curr Opin Cell Biol. 1997; 9: 233-239Crossref PubMed Scopus (173) Google Scholar, 3Meraz MA White JM Sheehan KCF Bach EA Rodig SJ Dighe AS Kaplan DH Riley JK Greenlund AC Campbell D Carver-Moore K DuBois RN Clark R Aguet M Schreiber RD Targeted disruption of the Stat-1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway.Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1383) Google Scholar, 4Walter MJ Look DC Tidwell RM Roswit WT Holtzman MJ Targeted inhibition of interferon-γ-dependent ICAM-1 expression using dominant-negative STAT-1.J Biol Chem. 1997; 272: 28582-28589Crossref PubMed Scopus (83) Google Scholar, 5O'Keefe GM Nguyen VT Ping Tang L Benveniste EN IFN-γ regulation of class II transactivator promoter IV in macrophages and microglia: involvement of the suppressors of cytokine signaling-1 protein.J Immunol. 2001; 166: 2260-2269PubMed Google Scholar Because of alternative splicing, STAT-1 exists in two forms: full-length STAT-1α and STAT-1β lacking 38 residues (including Ser-727) at the carboxy terminus. Only STAT-1α is able to activate transcription of IFN-γ-responsive genes.1Janeway CA Bottolmy K Signals and signs for lymphocyte responses.Cell. 1994; 76: 275-285Abstract Full Text PDF PubMed Scopus (766) Google Scholar, 2Horvath CM Darnell Jr, JE The state of the STATs: recent developments in the study of signal transduction to the nucleus.Curr Opin Cell Biol. 1997; 9: 233-239Crossref PubMed Scopus (173) Google ScholarBecause excessive IFN-γ stimulation might have deleterious consequences, it is not surprising that the IFN-γ/STAT-1 pathway is tightly regulated. Indeed it is known that some mechanisms are in place to modulate the cellular response to IFN-γ, and turn off the IFN-γ-activated signaling pathway.6Kovanen PE Leonard WJ Cytokine signaling: inhibitors keep cytokines in check.Curr Biol. 1999; 9: R899-R902Abstract Full Text Full Text PDF PubMed Google Scholar In this context, suppressor of cytokine signaling (SOCS)-1, a member of the SOCS family, plays a predominant role in suppressing IFN-γ-induced STAT-1 phosphorylation.7Alexander WS Starr R Fenner JE Scott CL Handman E Sprigg NS Corbin JE Cornish AL Darwiche R Owczarek CM Kay TWH Nicola NA Hertzog PJ Metcalf D Hilton DJ SOCS1 is a critical inhibitor of interferon γ signaling and prevents the potentially fatal neonatal actions of this cytokine.Cell. 1999; 98: 597-608Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar, 8Marine JC Topham DJ McKay C Wang D Parganas E Stravopodis D Yoshimura A Ihle JN SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality.Cell. 1999; 98: 609-616Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar SOCS-1 is an immediate early gene, and its transcripts, present at very low levels in immune cells, are rapidly up-regulated by signaling through IFN-γ/STAT-1 pathway.9Starr R Willson TA Viney EM Murray LJL Rayner JR Jenkins BJ Gonda TJ Alexander WS Metcalf D Nicola NA Hilton DJ A family of cytokine-inducible inhibitors of signaling.Nature. 1997; 387: 917-921Crossref PubMed Scopus (1792) Google Scholar Thus, SOCS-1 is part of a general negative feedback loop regulating IFN-γ action. More recently, it has however emerged that SOCS-1 protein is unstable and that cells can contain little or no SOCS-1 protein despite high RNA level.10Zhang J-G Farley A Nicholson SE Willson TA Zugaro LM Simpson RJ Moritz R Cary D Richardson R Hausmann G Kile BJ Kent SBH Alexander WS Metcalf D Hilton DJ Nicola NA Baca M The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation.Proc Natl Acad Sci USA. 1999; 96: 2071-2076Crossref PubMed Scopus (517) Google Scholar, 11Gregorief A Pyronnet S Sonenberg N Veillette A Regulation of SOCS-1 expression by translational repression.J Biol Chem. 2000; 275: 21596-21604Crossref PubMed Scopus (65) Google Scholar, 12Schulter G Boinska D Nieman-Seyde S-C Evidence for translational repression of the SOCS-1 open reading frame by an upstream open reading frame.Biochem Biophys Res Comm. 2000; 268: 255-261Crossref PubMed Scopus (40) Google ScholarCeliac disease (CD) is an enteropathy caused by dietary gluten in genetically susceptible individuals. The characteristic features of CD inflammation are villus atrophy, crypt cell hyperplasia, and increased number of intraepithelial lymphocytes.13Godkin A Jewell D The pathogenesis of celiac disease.Gastroenterology. 1998; 115: 206-210Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar The nature of CD pathogenesis remains unclear, but a large body of evidence indicates that CD4+ T cell-mediated hypersensitivity plays a major role in tissue injury in CD.14Schuppan D Current concept of celiac disease pathogenesis.Gastroenterology. 2000; 119: 234-242Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar Lamina propria CD4+ T cells are phenotypically activated and produce large amounts of IFN-γ when exposed to gluten.15Nilsen EM Jahnsen FL Lundin KEA Johansen F-E Fausa O Sollid LM Jahnsen J Scott H Brandtzaeg P Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease.Gastroenterology. 1998; 115: 551-563Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 16Breese EJ Kumar P Farthing MJG MacDonald TT Interleukin-2 and interferon-gamma producing cells in the lamina propria in coeliac disease.Dig Dis Sci. 1994; 39: 2243Crossref PubMed Scopus (28) Google Scholar In addition, gliadin-specific DQ2- and DQ8-restricted IFN-γ-producing T helper (Th) 1 CD4+ T cells have been derived from the intestinal lamina propria of CD patients.17Nilsen EM Lundin KEA Krajci P Scott H Sollid LM Brandtzaeg P Gluten specific, HLA-DQ restricted T cells from coeliac mucosa produce cytokines with Th1 or Th0 profile dominated by interferon γ.Gut. 1995; 37: 766-776Crossref PubMed Scopus (315) Google Scholar These observations, together with the demonstration that direct activation of lamina propria Th1 cells in explant cultures of human fetal gut produces villous atrophy and crypt cell hyperplasia, strongly support the role of IFN-γ in the CD immunopathology.18MacDonald TT Spencer J Evidence that activated mucosal T cells play a role in the pathogenesis of enteropathy in human small intestine.J Exp Med. 1988; 167: 1341-1349Crossref PubMed Scopus (330) Google Scholar, 19Lionetti P Breese E Braegger CP Murch SA Taylor J MacDonald TT T-cell activation can induce either mucosal destruction or adaptation in cultured human fetal small intestine.Gastroenterology. 1993; 105: 373-381Abstract PubMed Google Scholar, 20MacDonald TT Bajaj-Elliott M Pender SLF T cells orchestrate intestinal mucosal shape and integrity.Immunol Today. 1999; 20: 505-510Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar However, little is known about the signaling pathways and the factors that either positively or negatively regulate the IFN-γ driven biological effects in CD.Materials and MethodsPatients and ControlsBiopsy specimens from the distal duodenum of 32 patients with untreated CD (5 to 27 years of age) were obtained during upper gastrointestinal endoscopy. The histopathological diagnosis was based on typical mucosal lesions with crypt cell hyperplasia, villous atrophy, and increased number of intraepithelial lymphocytes. All untreated CD patients were positive for anti-endomysial (EMA) and anti-gliadin (AGA) antibodies. In all untreated CD patients, at least three biopsies were collected at the time of diagnosis. One specimen was used for routine histological examination, whereas the remaining mucosal samples were immediately frozen in liquid nitrogen and stored until tested. Biopsies were also obtained from eight treated CD patients (24 to 36 years of age), who were in clinical and histological remission, and negative for AGA/EMA antibodies. No patient had gluten-refractory disease. From these patients, four or more biopsies were collected: one was used histology and the remaining for organ culture. As control, two separate groups were considered. The first group of controls comprised four patients with nonspecific duodenitis and four with food enteropathy. Duodenal biopsies taken from these patients had normal mucosal architecture with long villi and short crypts and had a mild increase in nonspecific inflammatory cells. All of the disease control patients were EMA-negative. The second group was represented by age-matched normal patients (n = 38) who were under investigation for gastrointestinal symptoms, but had normal histology, no increase in inflammatory cells, and were EMA- and AGA-negative. This study received ethical approval from local committee (University Federico II, Naples, Italy).Organ Culture and Cell IsolationThe mucosal specimens were cultured as described elsewhere.21Fais S Maiuri L Pallone F De Vincenzi M De Ritis G Troncone R Auricchio S Gliadin induced changes in the expression of MHC-class II a by human small intestinal epithelium. Organ culture studies celiac disease mucosa.Gut. 1992; 33: 472-475Crossref PubMed Scopus (53) Google Scholar Briefly biopsies taken from patients with inactive CD were placed on iron grids with the mucosal face upwards in the central well of an organ culture dish in serum-free culture medium containing RPMI 1640 (Sigma, Milan, Italy) supplemented with 10% HL-1 (BioWhittaker, Wokingham, UK), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Life Technologies-GibcoBRL, Milan, Italy). Cultures were performed with or without the addition of 1 mg/ml of peptic-tryptic digest (Frazer III fraction) of gliadin (PT) (Sigma) in the presence or absence of the JAK/STAT inhibitor, tyrphostin B42 (TB42) (100 μmol/L; Inalco S.P.A., Milano, Italy) or dimethyl sulfoxide (100 μmol/L, Sigma). TB42 and dimethyl sulfoxide were preincubated for 4 hours before the addition of PT. The dishes were placed in a tight container with 95% O2/5% CO2 at 37°C, at 1 bar. Biopsies were snap-frozen after 24 hours and stored at −80°C until used. To examine if IFN-γ activates STAT-1 in human intestine, duodenal biopsies taken from normal controls were stimulated with rh-IFN-γ (final concentration, 100 ng/ml) (Peprotech EC Ltd., London, UK) for 1 to 4 hours, and then snap-frozen and stored at −80°C until used.Protein Extraction and Western Blot AnalysisWestern blot analysis was performed on whole mucosal duodenal samples from 12 active CD patients and 10 normal controls. Total proteins we prepared as previously described.22Monteleone G Trapasso F Parrello T Biancone L Stella A Iuliano R Luzza F Fusco A Pallone F Bioactive interleukin-18 expression is up-regulated in Crohn's disease.J Immunol. 1999; 163: 143-147PubMed Google Scholar For cytosolic and nuclear extracts, the method described by Schreiber and colleagues23Schreiber S Nikolaus S Hampe J Activation of nuclear factor kB in inflammatory bowel disease.Gut. 1998; 42: 477-484Crossref PubMed Scopus (619) Google Scholar was used with minor modifications. Briefly, snap-frozen biopsies were mechanically homogenized in liquid nitrogen, and cytosolic extracts collected in buffer A containing 10 mmol/L Hepes (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, and 0.2 mmol/L EGTA. Nuclear extracts were prepared by solubilizing the remaining nuclei in buffer C containing 20 mmol/L Hepes (pH 7.9), 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, and 10% glycerol. Both buffers were supplemented with 1 mmol/L dithiothreitol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mmol/L phenylmethane sulfonyl fluoride (all reagents were from Sigma).For the detection of IFN-γ, total proteins (100 μg/sample) were separated on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. IFN-γ was detected using a goat anti-human IFN-γ (1:300 final dilution) (Santa Cruz Biotechnology, Santa Cruz, CA) followed by a horseradish peroxidase-conjugated rabbit anti-goat IgG monoclonal antibody (DAKO, Cambridgeshire, UK) (final dilution, 1:2500). The reaction was detected with ECL Plus kit (Amersham Pharmaceuticals, Amersham, UK). To confirm equal loading and transfer of proteins, ponceau S (Sigma) staining was performed. In addition, after detection of IFN-γ, blots were stripped by incubation for 30 minutes at 50°C in stripping medium (2% SDS, 0.05 mol/L Tris, pH 6.8, 0.1 mmol/L β-mercaptoethanol) and analyzed for β-actin, as internal loading control, using a specific mouse anti-human β-actin antibody (1:5000 final dilution, Sigma), followed by a goat anti-mouse antibody conjugated to horseradish peroxidase (1:2500 dilution, DAKO).To investigate STAT-1 expression, cytosolic proteins (200 μg) were separated on a 8% SDS/PAGE gel, and analyzed for phosphorylated STAT-1 (p-STAT-1) using a mouse anti-human antibody that specifically recognizes STAT-1 phosphorylation on tyrosine 701 (1:1000 final dilution, Santa Cruz Biotechnology) or a rabbit anti-human antibody that specifically recognizes STAT-1 on serine 727 (1:5000 final dilution; Upstate Biotechnology, Lake Placid, NY). Rabbit anti-mouse or goat anti-rabbit antibody conjugated to horseradish peroxidase (1:2500 dilution, DAKO) was used as secondary antibody, and the reaction was developed with ECL-plus kit (Amersham Pharmaceuticals). After detection of p-STAT-1, blots were stripped and subsequently incubated with a rabbit anti-human STAT-1 polyclonal antibody (1:1000 final dilution, Santa Cruz Biotechnology) followed by a goat anti-rabbit antibody conjugated to horseradish peroxidase (1:2500 dilution, DAKO).To analyze the content of STAT-1 in the nuclear extracts, 10 μg of nuclear protein/sample were separated on an 8% SDS/PAGE gel and analyzed using the rabbit anti-human STAT-1 polyclonal antibody as indicated above. After detection of STAT-1, blots were stripped and subsequently incubated with a mouse anti-human histone-1 monoclonal antibody (1:300 final dilution, Santa Cruz Biotechnology) followed by a goat anti-mouse antibody conjugated to horseradish peroxidase (1:1500 dilution, DAKO).To investigate STAT-3 expression, cytosolic proteins (200 μg) were separated on an 8% SDS/PAGE gel, and analyzed for phosphorylated STAT-3 (p-STAT-3) using a mouse anti-human antibody that specifically recognizes STAT-3 tyrosine phosphorylation (1:500 final dilution, Santa Cruz Biotechnology). Goat anti-mouse antibody conjugated to horseradish peroxidase (1:2500 dilution, DAKO) was used as secondary antibody, and the reaction was developed with ECL-plus kit (Amersham Pharmaceuticals). After detection of p-STAT-3, blots were stripped and subsequently incubated with a rabbit anti-human STAT-3 polyclonal antibody (1:1000 final dilution, Santa Cruz Biotechnology) followed by a goat anti-rabbit antibody conjugated to horseradish peroxidase (1:2500 dilution, DAKO).For SOCS-1 protein analysis, cytosolic proteins (100 to 200 μg) were separated on a 12% SDS/PAGE gel and analyzed using a goat anti-human antibody that specifically recognizes SOCS-1 (1:300 final dilution, Santa Cruz Biotechnology). Rabbit anti-goat antibody conjugated to horseradish peroxidase (1:2500 dilution, DAKO) was used as secondary antibody, and the reaction was developed with ECL-plus kit (Amersham Pharmaceuticals). After detection of SOCS-1, blots were stripped and analyzed for β-actin as indicated above. SOCS-1 was also analyzed in immunoprecipitated proteins. For this purpose, 350 μg of cytosolic proteins were immunoprecipitated with an anti-SOCS-1 antibody (2 μg/ml, Santa Cruz Biotechnology) and analyzed by Western blotting. To confirm the specificity of the SOCS-1 band, proteins extracted from the same samples were immunoprecipitated using a control isotype antibody (DAKO). A different anti-SOCS-1 antibody (1:400 final dilution, Santa Cruz Biotechnology) followed by a horseradish peroxidase-conjugated rabbit anti-goat-antibody (1:5000 dilution, DAKO) was used. Immunoreactivity was visualized using an ECL kit (Amersham Pharmaceuticals).For SOCS-3 analysis, cytosolic proteins (100 μg) were separated on a 12% SDS/PAGE gel and analyzed using a goat anti-human antibody that specifically recognizes SOCS-3 (1:300 final dilution, Santa Cruz Biotechnology). Rabbit anti-goat antibody conjugated to horseradish peroxidase (1:2500 dilution, DAKO) was used as secondary antibody, and the reaction was developed with ECL-plus kit (Amersham Pharmaceuticals). After detection, blots were stripped and analyzed for β-actin as indicated above.ICAM-1 and B7-2 were examined in treated CD biopsies challenged in vitro with gliadin in the presence or absence of TB42. For this purpose total proteins (200 μg/sample) were incubated with a goat anti-human B7-2 or ICAM-1 (1:300 final dilution for both, Santa Cruz Biotechnology). Rabbit anti-goat antibody conjugated to horseradish peroxidase (1:2500 dilution, DAKO) was used as secondary antibody, and the reaction was developed with ECL-plus kit (Amersham Pharmaceuticals). Bands were quantified by densitometry and values expressed as arbitrary units.Electrophoretic Mobility Shift Assay (EMSA)EMSA was used to detect specific binding of activated STAT-1 to GAS-containing oligonucleotides from the promoter of the human FcyRI gene: 5′-GTATTTCCCAGAAAAGGAAC-3′. Nuclear protein-DNA-binding studies were performed for 20 minutes at room temperature in a 20-μl reaction volume containing 10 mmol/L Tris, 50 mmol/L KCl, 1 mmol/L dithiothreitol, 2.5% glycerol, 5 mmol/L MgCl2, 1 μg poly (dI-dC), (all of the reagents were from Sigma), 50 fmol biotin-labeled GAS-containing probe, and 14 μg of nuclear proteins. DNA probe was prepared by annealing the two consensus GAS oligonucleotides, which were labeled at the 3′ end with biotin using a commercially available kit (Pierce, Rockford, IL). The binding specificity was confirmed by incubating the nuclear protein samples with unlabeled GAS probe or nonspecific oligonucleotide of the interleukin (IL)-2 gene (IL2G), 5′-CACAACGCGTGAGCTCTCTAGAAAGCATCATCTCAACACTAACTTGATAATTAAGTGCCTCGAGCACA-3′) in 30-fold molar excess to compete binding. In antibody blocking assay, a monoclonal mouse anti-human STAT-1 (Santa Cruz Biotechnology) or control mouse IgG antibody (DAKO) (both used at a concentration of 2.5 μg) were incubated with the nuclear proteins for 45 minutes before adding the DNA probe. A 6% nondenaturing polyacrylamide gel was used for electrophoretic separation. After blotting to a membrane, labeled oligonucleotides were detected with a chemiluminescence EMSA kit (Pierce).RNA Extraction, cDNA Preparation, and Southern Blotting of Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) ProductsRNA was extracted using 1 ml of a monophasic solution of phenol and guanidine isothiocyanate (TRIzol; Life Technologies, Paisley, UK) and chloroform, followed by isopropanol (Sigma) precipitation. The integrity of RNA was checked by electrophoresis on a 1.5% agarose gel. A constant amount of RNA (150 ng/sample) was retrotranscribed into complementary cDNA, and 1 μl of cDNA/sample was then amplified using the following conditions: denaturation for 1 minute at 94°C, annealing for 1 minute at 55°C for both SOCS-1 and β-actin, and 58°C for SOCS-3, and extension for 1 minute and 15 seconds at 72°C as previously reported.22Monteleone G Trapasso F Parrello T Biancone L Stella A Iuliano R Luzza F Fusco A Pallone F Bioactive interleukin-18 expression is up-regulated in Crohn's disease.J Immunol. 1999; 163: 143-147PubMed Google Scholar, 24Monteleone G Biancone L Marasco R Morrone G Marasco O Luzza F Pallone F Interleukin 12 is expressed and actively released by Crohn's disease intestinal lamina propria mononuclear cells.Gastroenterology. 1997; 112: 1169-1174Abstract Full Text PDF PubMed Scopus (495) Google ScholarRNA expression for SOCS-1, SOCS-3, and β-actin was then assessed semiquantitatively by Southern blotting. In preliminary experiments we established the optimal number of cycles to obtain a PCR product within the linear phase of the amplification. For this purpose an equivalent amount of cDNA for sample was amplified using specific primers for β-actin (1 μl of cDNA for 19, 21, 23, and 25 cycles), SOCS-1 (1 μl of cDNA for 28, 30, 31, and 33 cycles), and SOCS-3 (1 μl of cDNA for 26, 28, 30, and 32 cycles). For Southern blot experiments cDNA samples were amplified with β-actin primers for 20 cycles, with SOCS-1 for 30 cycles, and with SOCS-3 for 26 cycles. The SOCS-1 and SOCS-3 primers were as follows: SOCS-1 forward: 5′-CACGCACTTCCGCACATTCC-3′ and SOCS-1 reverse: 5′-TCCAGCAGCTCGAAGAGGCA-3′; SOCS-3 forward: 5′-TCACCCACAGCAAGTTTCCCGC-3′ and SOCS-3 reverse: 5′-GTTGACGGTCTTCCGACAGAGAT-3′.β-actin primers have been published previously.22Monteleone G Trapasso F Parrello T Biancone L Stella A Iuliano R Luzza F Fusco A Pallone F Bioactive interleukin-18 expression is up-regulated in Crohn's disease.J Immunol. 1999; 163: 143-147PubMed Google Scholar Parallel experiments were performed using RNA as substrate for PCR assay to exclude the amplification of genomic DNA contaminating the RNA samples. PCR product specificity was confirmed by restriction analysis. The cDNA probes used in the Southern blotting were DNA fragments encoding the full-length PCR product. RT-PCR products were run on a 1% agarose gel and Southern blotting was performed according to a commercially available chemiluminescence detection kit (Amersham International). Bands were quantified by densitometry.ImmunohistochemistryTissue sections were cut, deparaffinized, and dehydrated through xylene and ethanol. For the purpose of antigen retrieval, the slides were incubated in the microwave oven for 20 minutes in 0.01 mol/L of citrate buffer, pH 6 (Sigma). To block endogenous peroxidase the slides were then incubated in 2% H2O2 for 20 minutes at room temperature. Incubation with the human monoclonal phospho-STAT-1 antibody (Santa Cruz Biotechnology) was performed at 4°C overnight. Primary antibody, used at a final concentration of 40 μg/ml, was omitted in sections used as negative control samples. After rinsing in Tris-buffered saline (Sigma), slides were incubated with an anti-mouse antibody conjugated to horseradish peroxidase (1:50 dilution, DAKO) for 40 minutes at room temperature. Immunoreactive cells were visualized by addition of diaminobenzidine (Sigma) as substrate and lightly counterstained with hematoxylin. Isotype control sections were prepared under identical immunohistochemical conditions, as described above, replacing the primary phospho-STAT-1 antibody with a purified, normal mouse IgG control antibody (DAKO). Tissue dehydration through graded alcohol and xylene was followed by mounting.ResultsActive STAT-1 in CDTo confirm that the CD lesion is associated with a marked expression of IFN-γ, Western blot analysis was performed using proteins extracted from duodenal mucosal samples of active CD patients and normal controls. In all CD patients and normal controls, anti-IFN-γ antibody detected a protein with a molecular size of 19 kd, co-migrating with recombinant human IFN-γ on SDS-PAGE (Figure 1). However, the intensity of the IFN-γ bands seen in CD was greater than that in normal controls (Figure 1), confirming previous reports showing that, in CD, the inflammatory response is associated with enhanced synthesis of IFN-γ.15Nilsen EM Jahnsen FL Lundin KEA Johansen F-E Fausa O Sollid LM Jahnsen J Scott H Brandtzaeg P Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease.Gastroenterology. 1998; 115: 551-563Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 16Breese EJ Kumar P Farthing MJG MacDonald TT Interleukin-2 and interferon-gamma producing cells in the lamina propria in coeliac disease.Dig Dis Sci. 1994; 39: 2243Crossref PubMed Scopus (28) Google ScholarBecause signals through the IFN-γ receptor lead to the STAT-1 activation, we next investigated if, in CD mucosa, there was enhanced activation of STAT-1. Proteins were prepared from biopsies taken from active CD patients and normal controls and separated by SDS-PAGE. Phosphorylation of STAT-1 (p-STAT-1) was then monitored by Western blotting analysis using an antibody that specifically recognize STAT-1 phosphorylation on tyrosine 701 (p-Tyr-STAT-1). As shown in Figure 2A (top blot), a strong band corresponding to p-Tyr-STAT-1α was seen in CD. In contrast, STAT-1 was weakly phosphorylated on the tyrosine 701 residue in normal controls (Figure 2A, top blot) and patients with duodenitis or food enteropathy (not shown). Although STAT-1 tyrosine phosphorylation is essential and sufficient to allow formation of DNA-binding STAT dimers, STAT-1 dimers will be transcriptionally inactive unless phosphorylated on serine 727.25Gamero AM Larner AC Signaling via the T cell antigen receptor induces phosphorylation of STAT-1 on serine 727.J Biol Chem. 2000; 275: 16574-16578Crossref PubMed Scopus (24) Google Scholar, 26Lafont V Decker T Cantrell D Antigen receptor signal transduction: activating and inhibitory antigen receptors regulate STAT-1 serine phosphorylation.Eur J Immunol. 2000; 30: 1851-1860Crossref PubMed Scopus (18) Google Scholar Therefore we then used an antibody, which specifically recognizes STAT-1 molecules phosphorylated on serine 727 (p-Ser-STAT-1) to monitor the phosphorylation of this critical STAT-1 residue. The data in Figure 2A, middle blot, show that mucosal samples taken from active CD patients exhibit a more pronounced p-Ser-STAT-1α in comparison to normal controls. Reprobing the blots with a pan-STAT-1 antibody demonstrated that approximately equal amounts of ST
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