Posttranslationally Modified Proteins as Mediators of Sustained Intestinal Inflammation
2006; Elsevier BV; Volume: 169; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2006.050713
ISSN1525-2191
AutoresMartin Andrassy, John C. Igwe, Frank Autschbach, Christian Volz, Andrew Remppis, Markus F. Neurath, Erwin Schleicher, Per M. Humpert, Thoralf Wendt, Birgit Liliensiek, Michael Morcos, Stephan Schiekofer, Kirsten Thiele, Jiang Chen, R. Kientsch-Engel, Ann‐Marie Schmidt, Wolfgang Stremmel, David M. Stern, Hugo A. Katus, Peter P. Nawroth, Angelika Bierhaus,
Tópico(s)Fatty Acid Research and Health
ResumoOxidative and carbonyl stress leads to generation of Nε-carboxymethyllysine-modified proteins (CML-mps), which are known to bind the receptor for advanced glycation end products (RAGE) and induce nuclear factor (NF)-κB-dependent proinflammatory gene expression. To determine the impact of CML-mps in vivo, RAGE-dependent sustained NF-κB activation was studied in resection gut specimens from patients with inflammatory bowel disease. Inflamed gut biopsy tissue demonstrated a significant up-regulation of RAGE and increased NF-κB activation. Protein extracts from the inflamed zones, but not from noninflamed resection borders, caused perpetuated NF-κB activation in cultured endothelial cells, which was mediated by CML-mps including CML-modified S100 proteins. The resulting NF-κB activation, lasting 5 days, was primarily inhibited by either depletion of CML-mps or by the addition of sRAGE, p44/42 and p38 MAPKinase-specific inhibitors. Consistently, CML-mps isolated from inflamed gut areas and rectally applied into mice caused NF-κB activation, increased proinflammatory gene expression, and histologically detectable inflammation in wild-type mice, but not in RAGE−/− mice. A comparable up-regulation of NF-κB and inflammation on rectal application of CML-mps was observed in IL-10−/− mice. Thus, CML-mps generated in inflammatory lesions have the capacity to elicit a RAGE-dependent intestinal inflammatory response. Oxidative and carbonyl stress leads to generation of Nε-carboxymethyllysine-modified proteins (CML-mps), which are known to bind the receptor for advanced glycation end products (RAGE) and induce nuclear factor (NF)-κB-dependent proinflammatory gene expression. To determine the impact of CML-mps in vivo, RAGE-dependent sustained NF-κB activation was studied in resection gut specimens from patients with inflammatory bowel disease. Inflamed gut biopsy tissue demonstrated a significant up-regulation of RAGE and increased NF-κB activation. Protein extracts from the inflamed zones, but not from noninflamed resection borders, caused perpetuated NF-κB activation in cultured endothelial cells, which was mediated by CML-mps including CML-modified S100 proteins. The resulting NF-κB activation, lasting 5 days, was primarily inhibited by either depletion of CML-mps or by the addition of sRAGE, p44/42 and p38 MAPKinase-specific inhibitors. Consistently, CML-mps isolated from inflamed gut areas and rectally applied into mice caused NF-κB activation, increased proinflammatory gene expression, and histologically detectable inflammation in wild-type mice, but not in RAGE−/− mice. A comparable up-regulation of NF-κB and inflammation on rectal application of CML-mps was observed in IL-10−/− mice. Thus, CML-mps generated in inflammatory lesions have the capacity to elicit a RAGE-dependent intestinal inflammatory response. Inflammatory bowel diseases (IBD) such as Crohn's disease (CD) and ulcerative colitis (UC) provide relevant model systems to study chronic inflammation mediated at least in part by sustained activation of the proinflammatory transcription factor nuclear factor (NF)-κB.1Ellis RD Limb GA Thompson RP Punchard NA NF kappa B in Crohn's disease.Biochem Soc Trans. 1997; 25: 178SCrossref PubMed Scopus (5) Google Scholar, 2Neurath MF Pettersson S Predominant role of NF-kappa B p65 in the pathogenesis of chronic intestinal inflammation.Immunobiology. 1997; 198: 91-98Crossref PubMed Scopus (77) Google Scholar, 3Neurath MF Pettersson S Meyer zum Buschenfelde KH Strober W Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice.Nat Med. 1996; 2: 998-1004Crossref PubMed Scopus (745) Google Scholar, 4Rogler G Brand K Vogl D Page S Hofmeister R Andus T Knuechel R Baeuerle PA Scholmerich J Gross V Nuclear factor kappaB is activated in macrophages and epithelial cells of inflamed intestinal mucosa.Gastroenterology. 1998; 115: 357-369Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 5Schreiber S Nikolaus S Hampe J Activation of nuclear factor kappa B inflammatory bowel disease.Gut. 1998; 42: 477-484Crossref PubMed Scopus (619) Google Scholar, 6Thiele K Bierhaus A Autschbach F Hofmann M Stremmel W Thiele H Ziegler R Nawroth PP Cell specific effects of glucocorticoid treatment on the NF-kappaBp65/IkappaBalpha system in patients with Crohn's disease.Gut. 1999; 45: 693-704Crossref PubMed Scopus (77) Google Scholar Because of the strong positive correlation between NF-κB activation in gut tissue and the clinical course of CD,4Rogler G Brand K Vogl D Page S Hofmeister R Andus T Knuechel R Baeuerle PA Scholmerich J Gross V Nuclear factor kappaB is activated in macrophages and epithelial cells of inflamed intestinal mucosa.Gastroenterology. 1998; 115: 357-369Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 5Schreiber S Nikolaus S Hampe J Activation of nuclear factor kappa B inflammatory bowel disease.Gut. 1998; 42: 477-484Crossref PubMed Scopus (619) Google Scholar, 6Thiele K Bierhaus A Autschbach F Hofmann M Stremmel W Thiele H Ziegler R Nawroth PP Cell specific effects of glucocorticoid treatment on the NF-kappaBp65/IkappaBalpha system in patients with Crohn's disease.Gut. 1999; 45: 693-704Crossref PubMed Scopus (77) Google Scholar NF-κB is regarded as one of the main targets of anti-inflammatory therapies in human chronic active IBD.7Hollenbach E Vieth M Roessner A Neumann M Malfertheiner P Naumann M Inhibition of RICK/nuclear factor-kappaB and p38 signaling attenuates the inflammatory response in a murine model of Crohn disease.J Biol Chem. 2005; 280: 14981-14988Crossref PubMed Scopus (131) Google ScholarA critical question, however, concerns mechanisms capable of converting a pulse of proinflammatory cytokines and the transitory burst of reactive oxygen species and arachidonic acid metabolites1Ellis RD Limb GA Thompson RP Punchard NA NF kappa B in Crohn's disease.Biochem Soc Trans. 1997; 25: 178SCrossref PubMed Scopus (5) Google Scholar, 2Neurath MF Pettersson S Predominant role of NF-kappa B p65 in the pathogenesis of chronic intestinal inflammation.Immunobiology. 1997; 198: 91-98Crossref PubMed Scopus (77) Google Scholar, 3Neurath MF Pettersson S Meyer zum Buschenfelde KH Strober W Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappa B abrogates established experimental colitis in mice.Nat Med. 1996; 2: 998-1004Crossref PubMed Scopus (745) Google Scholar, 4Rogler G Brand K Vogl D Page S Hofmeister R Andus T Knuechel R Baeuerle PA Scholmerich J Gross V Nuclear factor kappaB is activated in macrophages and epithelial cells of inflamed intestinal mucosa.Gastroenterology. 1998; 115: 357-369Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar, 5Schreiber S Nikolaus S Hampe J Activation of nuclear factor kappa B inflammatory bowel disease.Gut. 1998; 42: 477-484Crossref PubMed Scopus (619) Google Scholar, 6Thiele K Bierhaus A Autschbach F Hofmann M Stremmel W Thiele H Ziegler R Nawroth PP Cell specific effects of glucocorticoid treatment on the NF-kappaBp65/IkappaBalpha system in patients with Crohn's disease.Gut. 1999; 45: 693-704Crossref PubMed Scopus (77) Google Scholar, 8Bierhaus A Nawroth PP Modulation of the vascular endothelium during infection—the role of NF-kappa B activation.Contrib Microbiol. 2003; 10: 86-105Crossref PubMed Google Scholar, 9Mercurio F Manning AM NF-kappaB as a primary regulator of the stress response.Oncogene. 1999; 18: 6163-6171Crossref PubMed Scopus (359) Google Scholar into a sustained inflammatory stimulus resulting in long-lasting NF-κB activation. 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One unique feature of RAGE-mediated NF-κB activation is the prolonged time course that appears to overwhelm endogenous autoregulatory feedback inhibition loops and to induce perpetuated NF-κB activation.25Bierhaus A Schiekofer S Schwaninger M Andrassy M Humpert PM Chen J Hong M Luther T Henle T Kloting I Morcos M Hofmann M Tritschler H Weigle B Kasper M Smith M Perry G Schmidt AM Stern DM Haring HU Schleicher E Nawroth PP Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB.Diabetes. 2001; 50: 2792-2808Crossref PubMed Scopus (722) Google Scholar Because, in turn, RAGE expression is induced by NF-κB,26Li J Schmidt AM Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products.J Biol Chem. 1997; 272: 16498-16506Crossref PubMed Scopus (444) Google Scholar sustained activation of NF-κB results in up-regulation of the receptor and further ensures maintenance and amplification of the signal. In the study presented here, we demonstrate for the first time that post-translationally modified proteins such as CML-mps, which also include S100A8, S100A9, and S100A8/9, are direct mediators of inflammation in predisposed tissue and are able to convert a short-lasting cytokine-driven initiation into a longer-lasting sustained inflammation.Materials and MethodsPatientsSix patients with CD (matched for CDAI and characterized as previously described in detail6Thiele K Bierhaus A Autschbach F Hofmann M Stremmel W Thiele H Ziegler R Nawroth PP Cell specific effects of glucocorticoid treatment on the NF-kappaBp65/IkappaBalpha system in patients with Crohn's disease.Gut. 1999; 45: 693-704Crossref PubMed Scopus (77) Google Scholar) and five patients with UC were included in the study; clinical characteristics are given in Table 1. Because the part of the inflamed intestine removed during surgery was different in different patients, resection border and inflamed area of the same patient were compared with each other throughout the study. All patients included were under medical treatment in the Department of Visceral Surgery in Bruchsal, Germany, and gave informed consent for study participation. The study was approved by the ethics committee of the University of Heidelberg according to the guidelines of the Helsinki Declaration.Table 1Clinical Characteristics of the Patients StudiedCrohn's disease (CD)Ulcerative colitis (UC)Sex (M/F)3/33/2Age34.2 ± 7.238.2 ± 4.2Duration of disease (years)8.5 ± 1.46.2 ± 2.8Hemoglobin (g/dl)12.4 ± 2.611.4 ± 2.4Leucocytes (×109/L)14.3 ± 2.313.7 ± 1.9Time of glucocorticoid therapy (months)3.4 ± 1.21.4 ± 0.8CDAI289 ± 35Van Hees index190 ± 25 Open table in a new tab Reagents[γ-32P]ATP (3000 Ci/mmol at 10 Ci/ml); Hybond-N nylon filter, enhanced chemiluminescence (ECL)-nitrocellulose membranes, ECL detection reagents, and Hyperfilm X-ray-films were obtained from Amersham, Braunschweig, Germany. Poly(dI/dC) was from Pharmacia, Freiburg, Germany. Monoclonal anti-NF-κBp65 antibodies, specific for activated NF-κBp65, were obtained from Boehringer Mannheim, Mannheim, Germany. Anti-p65-antibodies (no. sc-109) were obtained from Santa Cruz Inc., Heidelberg, Germany. Anti-CML antibodies were produced and characterized as described recently.13Schleicher ED Wagner E Nerlich AG Increased accumulation of the glycoxidation product N(epsilon)-(carboxymethyl)lysine in human tissues in diabetes and aging.J Clin Invest. 1997; 99: 457-468Crossref PubMed Scopus (658) Google Scholar Antibodies for S100A8/9, S100A8, S100A9, and S100A1 were purchased from BMA, Basel, Switzerland. PD 98059 and SB203580 were purchased from Sigma-Aldrich, Deisenhofen, Germany, and MG132 was from Biomol (Darmstadt, Germany).CML-Enzyme-Linked Immunosorbent Assay (ELISA)CML detection by ELISA (Roche Diagnostics, Penzberg, Germany) was performed according to the manufacturer's instructions as recently described in detail.27Morcos M Sayed AA Bierhaus A Yard B Waldherr R Merz W Kloeting I Schleicher E Mentz S Abd el Baki RF Tritschler H Kasper M Schwenger V Hamann A Dugi KA Schmidt AM Stern D Ziegler R Haering HU Andrassy M van der Woude F Nawroth PP Activation of tubular epithelial cells in diabetic nephropathy.Diabetes. 2002; 51: 3532-3544Crossref PubMed Scopus (146) Google ScholarPreparation of Tissues and ImmunohistochemistryPreparation and immunohistochemistry of the gut samples were performed as previously described.6Thiele K Bierhaus A Autschbach F Hofmann M Stremmel W Thiele H Ziegler R Nawroth PP Cell specific effects of glucocorticoid treatment on the NF-kappaBp65/IkappaBalpha system in patients with Crohn's disease.Gut. 1999; 45: 693-704Crossref PubMed Scopus (77) Google Scholar, 28Autschbach F Braunstein J Helmke B Zuna I Schurmann G Niemir ZI Wallich R Otto HF Meuer SC In situ expression of interleukin-10 in noninflamed human gut and in inflammatory bowel disease.Am J Pathol. 1998; 153: 121-130Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar Resection gut specimens of patients with CD and UC were taken during operation and snap-frozen in liquid nitrogen. Cryostat sections (5 μm) were mounted and fixed in 2% paraformaldehyde (Merck, Darmstadt, Germany). Acetone-treated slides were postfixed with 2% paraformaldehyde before this step. For detection of RAGE, sections were washed in Earl's balanced salt solution (Gibco BRL, Eggenstein, Germany) plus 0.01 mol/L HEPES and 0.1% Saponin (Riedel deHaen, Seelze, Germany).28Autschbach F Braunstein J Helmke B Zuna I Schurmann G Niemir ZI Wallich R Otto HF Meuer SC In situ expression of interleukin-10 in noninflamed human gut and in inflammatory bowel disease.Am J Pathol. 1998; 153: 121-130Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar Anti-human RAGE antibodies were diluted 1:50 and incubated overnight in a humidified chamber in the presence of 2.5 mg/ml human immunoglobulins (Gamma-venin, Marburg, Germany) to reduce nonspecific background.28Autschbach F Braunstein J Helmke B Zuna I Schurmann G Niemir ZI Wallich R Otto HF Meuer SC In situ expression of interleukin-10 in noninflamed human gut and in inflammatory bowel disease.Am J Pathol. 1998; 153: 121-130Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar After washing in Earl's balanced salt solution/HEPES/Saponin-buffer (2 × 3 minutes) and incubating with 5% inactivated goat serum for 20 minutes, the biotinylated secondary antibody (goat anti-rabbit, 1:20; Sigma, Deisenhofen, Germany) was added for 30 minutes, followed by a 30-minute incubation with streptavidin complexed with alkaline phosphatase (DAKO, Copenhagen, Denmark).29Hofmann MA Schiekofer S Isermann B Kanitz M Henkels M Joswig M Treusch A Morcos M Weiss T Borcea V Abdel Khalek AK Amiral J Tritschler H Ritz E Wahl P Ziegler R Bierhaus A Nawroth PP Peripheral blood mononuclear cells isolated from patients with diabetic nephropathy show increased activation of the oxidative-stress sensitive transcription factor NF-kappaB.Diabetologia. 1999; 42: 222-232Crossref PubMed Scopus (180) Google Scholar After subsequent washes with Earl's balanced salt solution/HEPES/Saponin (3 × 2 minutes) and a final wash in distilled water, the color reaction was developed using naphthol AS-bi phosphate and new fuchsin as chromogen. For detection of NF-κBp65, a monoclonal mouse antibody for activated NF-κBp65 (1:10 diluted in Tris-buffered saline, pH 7.4, containing 0.2% bovine serum albumin) was applied to the cryostat sections for 90 minutes at room temperature followed by washing in Tris-buffered saline/0.2% bovine serum albumin (2 × 3 minutes). Thereafter, a biotin-sheep anti-mouse antibody (1:100; Amersham, Freiburg, Germany) was added for 30 minutes, before detection was performed using the alkaline phosphatase anti-alkaline phosphatase immunostaining method as above. After counterstaining with hematoxylin, sections were mounted in glycerol/gelatin. The immunohistochemical results were evaluated according to a score depicted by Thiele and colleagues.6Thiele K Bierhaus A Autschbach F Hofmann M Stremmel W Thiele H Ziegler R Nawroth PP Cell specific effects of glucocorticoid treatment on the NF-kappaBp65/IkappaBalpha system in patients with Crohn's disease.Gut. 1999; 45: 693-704Crossref PubMed Scopus (77) Google Scholar In brief, the score refers to the staining result of mononuclear cells and endothelial cells. Zero means no positive cells and no staining of endothelia, whereas 8 means 81 to 100% positive cells with a strong staining, up to 81 to 100% of vessel circumference stained strongly positive.6Thiele K Bierhaus A Autschbach F Hofmann M Stremmel W Thiele H Ziegler R Nawroth PP Cell specific effects of glucocorticoid treatment on the NF-kappaBp65/IkappaBalpha system in patients with Crohn's disease.Gut. 1999; 45: 693-704Crossref PubMed Scopus (77) Google Scholar One investigator (K.T.) blinded to the clinical diagnosis analyzed coded slides. Expression of NF-κBp65 and RAGE was determined on consecutive serial sections in each case. In each slide, the number of positive mononuclear cells was determined in at least five visual fields (magnification, ×160) in five different representative areas of the lamina propria as well as in the submucosa. For determination of the endothelial expression, 40 vessels in the lamina propria and the submucosa were analyzed in each case. Median values of the respective results were obtained for statistical evaluation.Preparation of Cell Extracts for Electrophoretic Mobility Shift Assays (EMSAs)Frozen tissue was homogenized mechanically under liquid nitrogen using a mortar and pestle and transferred into ice-cold buffer A (10 mmol/L Hepes/KOH, pH 7.9, at 4°C, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol, 0.2 mmol/L phenylmethyl sulfonyl fluoride, and 0.6% Nonidet-P40). Insoluble material was removed by centrifugation (30 seconds at 2000 rpm, 4°C), and the supernatant was incubated on ice for 10 minutes before being centrifuged for 5 minutes at 8000 rpm, 4°C. The supernatant was discarded, and the nuclear pellet was resuspended in 100 μl of buffer B (25% glycerol, 20 mmol/L HEPES/KOH, pH 7.9, at 4°C, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L ethylenediaminetetraacetic acid, 0.5 mmol/L dithiothreitol, 0.2 mmol/L phenylmethyl sulfonyl fluoride, 2 mmol/L benzamidine, and 5 mg/ml leupeptin) and incubated on ice for 20 minutes. Cellular debris was removed by 2 minutes of centrifugation at 14,000 rpm, 4°C, and the supernatant was quick-frozen at −80°C.6Thiele K Bierhaus A Autschbach F Hofmann M Stremmel W Thiele H Ziegler R Nawroth PP Cell specific effects of glucocorticoid treatment on the NF-kappaBp65/IkappaBalpha system in patients with Crohn's disease.Gut. 1999; 45: 693-704Crossref PubMed Scopus (77) Google Scholar, 25Bierhaus A Schiekofer S Schwaninger M Andrassy M Humpert PM Chen J Hong M Luther T Henle T Kloting I Morcos M Hofmann M Tritschler H Weigle B Kasper M Smith M Perry G Schmidt AM Stern DM Haring HU Schleicher E Nawroth PP Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB.Diabetes. 2001; 50: 2792-2808Crossref PubMed Scopus (722) Google ScholarEMSAsNuclear proteins were harvested, and 10 μg of nuclear proteins were assayed for NF-κB binding activity using radioactive labeled oligonucleotides for the defined NF-κB consensus sequence (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) at ∼50,000 cpm (Cerénkov). Binding reaction was performed for 20 minutes at room temperature in 10 mmol/L HEPES, pH 7.5, 0.5 mmol/L ethylenediaminetetraacetic acid, 100 mmol/L KCl, 2 mmol/L dithiothreitol, 2% glycerol, 4% Ficoll, 0.25% Nonidet P-40, 1 mg/ml bovine serum albumin, and 0.1 μg/μl poly(dI/dC) as previously described.25Bierhaus A Schiekofer S Schwaninger M Andrassy M Humpert PM Chen J Hong M Luther T Henle T Kloting I Morcos M Hofmann M Tritschler H Weigle B Kasper M Smith M Perry G Schmidt AM Stern DM Haring HU Schleicher E Nawroth PP Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB.Diabetes. 2001; 50: 2792-2808Crossref PubMed Scopus (722) Google Scholar Protein-DNA complexes were separated from the unbound DNA by electrophoresis through 5% native polyacrylamide gels containing 2.5% glycerol and 0.5× TBE. Gels were dried under vacuum and exposed for 48 to 64 hours to Amersham Hyperfilms (Amersham) at −80°C with intensifying screens. Specificity of binding was ascertained by competition with a 160-fold molar excess of unlabeled consensus oligonucleotides.25Bierhaus A Schiekofer S Schwaninger M Andrassy M Humpert PM Chen J Hong M Luther T Henle T Kloting I Morcos M Hofmann M Tritschler H Weigle B Kasper M Smith M Perry G Schmidt AM Stern DM Haring HU Schleicher E Nawroth PP Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB.Diabetes. 2001; 50: 2792-2808Crossref PubMed Scopus (722) Google Scholar Each EMSA signal was quantified by densitometry, and the mean ± SD is given.6Thiele K Bierhaus A Autschbach F Hofmann M Stremmel W Thiele H Ziegler R Nawroth PP Cell specific effects of glucocorticoid treatment on the NF-kappaBp65/IkappaBalpha system in patients with Crohn's disease.Gut. 1999; 45: 693-704Crossref PubMed Scopus (77) Google Scholar, 29Hofmann MA Schiekofer S Isermann B Kanitz M Henkels M Joswig M Treusch A Morcos M Weiss T Borcea V Abdel Khalek AK Amiral J Tritschler H Ritz E Wahl P Ziegler R Bierhaus A Nawroth PP Peripheral blood mononuclear cells isolated from patients with diabetic nephropathy show increased activation of the oxidative-stress sensitive transcription factor NF-kappaB.Diabetologia. 1999; 42: 222-232Crossref PubMed Scopus (180) Google ScholarWestern Blot AnalysisCytoplasmic and nuclear fractions were prepared as previously described.6Thiele K Bierhaus A Autschbach F Hofmann M Stremmel W Thiele H Ziegler R Nawroth PP Cell specific effects of glucocorticoid treatment on the NF-kappaBp65/IkappaBalpha system in patients with Crohn's disease.Gut. 1999; 45: 693-704Crossref PubMed Scopus (77) Google Scholar, 25Bierhaus A Schiekofer S Schwaninger M Andrassy M Humpert PM Chen J Hong M Luther T Henle T Kloting I Morcos M Hofmann M Tritschler H Weigle B Kasper M Smith M Perry G Schmidt AM Stern DM Haring HU Schleicher E Nawroth PP Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB.Diabetes. 2001; 50: 2792-2808Crossref PubMed Scopus (722) Google Scholar Twenty μg of cytoplasmic extracts or 10 μg of nuclear extracts were separated onto 10 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, followed by electroblotting to ECL-nitrocellulose (CML-mps) or polyvinylidene difluoride (S100 proteins) membranes, respectively. Membranes were incubated with primary antibodies for CML (1:8000), S100A1, S100A8, and S100A9 (1:10,000) for 60 minutes at room temperature. After washing (2 × 7 minutes in Tris-buffered saline, 0.05% Tween), the secondary antibody (horseradish peroxidase-coupled rabbit IgG, 1:2000) was added, and incubation was continued for 30 minutes at room temperature. Membranes were washed 3 × 15 minutes as above followed by a final 5-minute wash in Tris-buffered saline. Immunoreactive proteins were detected with the ECL-Western blot system and subsequent autoradiography for 2 to 10 minutes.Immunoprecipitation of S100A8 and S100A8/9 ProteinsImmunoprecipitation was performed using protein A/G PLUS agarose immunoprecipitation reagent according to the guidelines of Santa Cruz Biotechnology, Inc., Santa Cruz, CA. In brief, 400 μg of total gut protein lysates derived from representative patients with CD and colitis were incubated with 1 μg of S100A8 or 1 μg of S100A8/9-specific antibodies respectively for 2 hours at 4°C. The resulting S100A8 and S100A8/9 immunoprecipitates were incubated with 20 μl of resuspended protein A/G PLUS-agarose for an additional 2 hours at 4°C. Immunoprecipitates were collected by careful centrifugation (1000 × g) for 5 minutes at 4°C and pellet was washed four times with phosphate-buffered saline before resuspension in electrophoresis buffer.Elution of CML-mps and S100 Proteins from TissuesIsolation of CML-mps and S100 proteins was performed as previously described.30Quehenberger P Bierhaus A Fasching P Muellner C Klevesath M Hong M Stier G Sattler M Schleicher E Speiser W Nawroth PP Endothelin 1 transcription is controlled by nuclear factor-kappaB in AGE-stimulated cult
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