Enteric Glial-Derived S100B Protein Stimulates Nitric Oxide Production in Celiac Disease
2007; Elsevier BV; Volume: 133; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2007.06.009
ISSN1528-0012
AutoresGiuseppe Esposito, Carla Cirillo, Giovanni Sarnelli, Daniele De Filippis, Francesco Paolo D’Armiento, Alba Rocco, Gerardo Nardone, Raffaella Petruzzelli, Michela Grosso, Paola Izzo, Teresa Iuvone, Rosario Cuomo,
Tópico(s)Vitamin C and Antioxidants Research
ResumoBackground & Aims: Enteric glia participates to the homeostasis of the gastrointestinal tract. In the central nervous system, increased expression of astroglial-derived S100B protein has been associated with the onset and maintaining of inflammation. The role of enteric glial-derived S100B protein in gastrointestinal inflammation has never been investigated in humans. In this study, we evaluated the expression of S100B and its relationship with nitric oxide production in celiac disease. Methods: Duodenal biopsy specimens from untreated and on gluten-free diet patients with celiac disease and controls were respectively processed for S100B and inducible nitric oxide synthase (iNOS) protein expression and nitrite production. To evaluate the direct involvement of S100B in the inflammation, control biopsy specimens were exposed to exogenous S100B, and iNOS protein expression and nitrite production were measured. We also tested gliadin induction of S100B-dependent inflammation in cultured biopsy specimens deriving from on gluten-free diet patients in the absence or presence of the specific S100B antibody. Results: S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production were significantly increased in untreated patients but not in on gluten-free diet patients vs controls. Addition of S100B to control biopsy specimens resulted in a significant increase of iNOS protein expression and nitrite production. In celiac disease patients but not in controls biopsy specimens, gliadin challenge significantly increased S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production, but these effects were completely inhibited by S100B antibody. Conclusions: Enteric glial-derived S100B is increased in the duodenum of patients with celiac disease and plays a role in nitric oxide production. Background & Aims: Enteric glia participates to the homeostasis of the gastrointestinal tract. In the central nervous system, increased expression of astroglial-derived S100B protein has been associated with the onset and maintaining of inflammation. The role of enteric glial-derived S100B protein in gastrointestinal inflammation has never been investigated in humans. In this study, we evaluated the expression of S100B and its relationship with nitric oxide production in celiac disease. Methods: Duodenal biopsy specimens from untreated and on gluten-free diet patients with celiac disease and controls were respectively processed for S100B and inducible nitric oxide synthase (iNOS) protein expression and nitrite production. To evaluate the direct involvement of S100B in the inflammation, control biopsy specimens were exposed to exogenous S100B, and iNOS protein expression and nitrite production were measured. We also tested gliadin induction of S100B-dependent inflammation in cultured biopsy specimens deriving from on gluten-free diet patients in the absence or presence of the specific S100B antibody. Results: S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production were significantly increased in untreated patients but not in on gluten-free diet patients vs controls. Addition of S100B to control biopsy specimens resulted in a significant increase of iNOS protein expression and nitrite production. In celiac disease patients but not in controls biopsy specimens, gliadin challenge significantly increased S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production, but these effects were completely inhibited by S100B antibody. Conclusions: Enteric glial-derived S100B is increased in the duodenum of patients with celiac disease and plays a role in nitric oxide production. Enteric glial cells (EGC) have been fully reevaluated in recent years as a fundamental cell type involved in the maintenance of gut microenvironment homeostasis.1Giaroni C. De Ponti F. Casentino M. et al.Plasticity in the enteric nervous system.Gastroenterology. 1999; 117: 1438-1458Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar Similarly to their equivalent in the central nervous system (CNS), EGC release a wide range of neurotrophic factors accounting for development, survival, and differentiation of many peripheral neurons.2Sariola H. Saarma M. Novel functions and signalling pathways for GDNF.J Cell Sci. 2003; 116: 3855-3862Crossref PubMed Scopus (514) Google Scholar In addition, EGC are activated by inflammatory insults and may actively contribute to an inflammatory condition via antigen presentation and cytokine synthesis.3Ruhl A. Nasser Y. Sharkey K.A. Enteric glia.Neurogastroenterol Motil. 2004; 16: 44-49Crossref PubMed Scopus (118) Google Scholar EGC represent, thus, a very important link between the nervous and immune systems of the gut, playing a pivotal role in maintaining the integrity of the mucosal barrier.3Ruhl A. Nasser Y. Sharkey K.A. Enteric glia.Neurogastroenterol Motil. 2004; 16: 44-49Crossref PubMed Scopus (118) Google Scholar, 4Bush T.G. Savidge T.C. Freeman T.C. et al.Fulminant jejuno-ileitis following ablation of enteric glia in adult transgenic mice.Cell. 1998; 93: 189-201Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar During chronic gut inflammation, EGC proliferate and release neurotrophins, growth factors, and proinflammatory cytokines, which, in turn, may amplify the immune response.5Cornet A. Savidge T.C. Cabarrocas J. et al.Enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in Crohn's disease?.Proc Natl Acad Sci U S A. 2001; 98: 13306-13311Crossref PubMed Scopus (270) Google Scholar One of the most interesting neurotrophins released by astroglial cells is the S100B protein, a Ca+2/Zn+2-binding protein localized in the cytoplasm and/or nucleus of a wide range of both nervous and nonnervous tissues.6Haimoto H. Hosoda S. Kato K. Differential distribution of immunoreactive S100 α and S100 β proteins in normal nonnervous human tissue.Lab Invest. 1987; 57: 489-498PubMed Google Scholar, 7Zimmer D.B. Van Eldik L.J. Tissue distribution of rat S100 α and S100 β and S100 binding proteins.Am J Physiol. 1987; 252: 285-289PubMed Google Scholar Despite its neurotrophic role, an aberrant production of S100B has been reported to play a deleterious role in different neuropathologies of the CNS.8Van Eldik L.J. Wainwright M.S. The Janus face of glial-derived S100B: beneficial and detrimental functions in the brain.Restor Neurol Neurosci. 2003; 21: 97-108PubMed Google Scholar In the brain, S100B may induce the expression of specific proinflammatory transcription factors9Goncalves D.S. Lenz G. Karl J. et al.Extracellular S100B protein modulates ERK in astrocyte cultures.Neuroreport. 2000; 11: 807-809Crossref PubMed Scopus (64) Google Scholar, 10Lam A.G. Koppal T. Akama K.T. et al.Mechanism of glial activation by S100B: involvement of the transcription factor NF-κB.Neurobiol Aging. 2001; 22: 765-772Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar inducing the synthesis and release of nitric oxide (NO) in an autocrine manner by astroglial cells.11Hu J. Ferreira A. Van Eldik L.J. S100β induces neuronal cell death through nitric oxide release from astrocytes.J Neurochem. 1997; 69: 2294-2301Crossref PubMed Scopus (321) Google Scholar In the gut, S100B is exclusively localized in enteroglial cells12Rhul A. Glial cells in the gut.Neurogastroenterol Motil. 2005; 17: 777-790Crossref PubMed Scopus (204) Google Scholar, 13Ferri G.L. Probert L. Cocchia D. et al.Evidence for the presence of S-100 protein in the glial component of the human enteric nervous system.Nature. 1982; 297: 409-412Crossref PubMed Scopus (193) Google Scholar and is considered a specific marker of enteric glia.14Neunlist M. Aubert P. Bonnaud S. et al.Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGF-β1-dependent pathway.Am J Physiol Gastrointest Liver Physiol. 2007; 292: 231-241Crossref PubMed Scopus (142) Google Scholar Although S100B, together with other S100 proteins, have been related to the development and progression of colon cancer,15Bronckart Y. Decaestecker C. Nagy N. et al.Development and progression of malignancy in human colon tissue are correlated with expression of specific Ca(2+)-binding S100 protein.Histol Histopatol. 2001; 16: 707-712PubMed Google Scholar there are no data about the role exerted by this protein in human chronic gut inflammation. Celiac disease (CD) represents a paradigmatic chronic enteropathy16Dewar D.H. Ciclitira P.J. Clinical features and dignosis of celiac disease.Gastroenterology. 2005; 128: 19-24Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar characterized by a complex inflammatory immunoreaction with release of proinflammatory cytokines and NO.17Murray I.A. Daniels I. Coupland K. et al.Increased activity and expression of iNOS in human duodenal enterocytes from patients with celiac disease.Am J Physiol Gastrointest Liver Physiol. 2002; 283: 319-326Crossref Scopus (47) Google Scholar On the basis of this background, in the present study, we evaluated the expression of S100B and its relationship with NO-dependent inflammation in the duodenal mucosa of CD patients. Duodenal biopsy specimens were taken during upper gastrointestinal endoscopy in 22 patients with untreated CD (mean age, 34 years; range, 23–45) serologically diagnosed on the basis of immunoglobulin (Ig)A endomysial, gliadin antibodies, and transglutaminase antibodies positivity and in 21 CD patients on a gluten-free diet (GFD-CD) (mean age, 35 years; range 23–47). Twenty-three subjects (mean age, 45 years; range, 23–67) with dyspeptic symptoms served as controls. We obtained informed consent from all the participants and approval from the Ethics Committee of the Federico II University of Naples. Diagnosis of CD was confirmed by duodenal histology according to the Marsh criteria.18Marsh M.N. Bjarnason I. Shaw J. et al.Studies of intestinal lymphoid tissue XIV—HLA status, mucosal morphology, permeability and epithelial lymphocyte populations in first-degree relatives of patients with coeliac disease.Gut. 1990; 31: 111-114Crossref PubMed Scopus (123) Google Scholar The degree of mucosal inflammation was further characterized by myeloperoxidase assay, which was significantly increased (127% ± 8%; P < .01) in CD patients compared with GFD-CD and controls. According to Coeffier et al,19Coeffier M. Miralles-Barrachina O. Le Pessot F. et al.Influence of glutamine on cytokine production by human gut in vitro.Cytokine. 2001; 13: 148-154Crossref PubMed Scopus (105) Google Scholar whole biopsy specimens from controls, CD, and GFD-CD patients, were placed in 24-well plates for 24 hours and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mg/mL gentamycin (Biowhittaker, Milan, Italy) at 37°C in 5% CO2/95% air. The release of S100B and nitrite levels, using the Griess reagent,20Di Rosa M. Radomski M. Carnuccio R. et al.Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages.Biochem Biophys Res Commun. 1990; 172: 1246-1252Crossref PubMed Scopus (437) Google Scholar were measured in the supernatant on the basis of different experimental conditions. For S100B immunohistochemistry, duodenal specimens deriving from controls, CD, and GFD-CD patients were fixed in buffered formalin, embedded in paraffin, and cut into 4-μm-thick serial sections. Sections were stained with the primary S100B antibody (1:200; NeoMarker, Fremont, CA) and counterstained with H&E at room temperature. After 3, 5-minute washes, the secondary antibody was added, and the samples were incubated at room temperature for 20 minutes. The streptavidin-horseradish peroxidase detection system (Chemicon Int, Temecula, CA) was added, and samples were incubated at room temperature. After 3, 5-minute washes, 50 μL chromogen was added and the reaction terminated after 1 minute in water. Appropriate negative controls were performed by omitting primary antibody. Total RNA was isolated from homogenized tissue using Trizol reagent (Invitrogen, Milan, Italy) according to the manufacturer's instructions. One microgram of total RNA was used to generate a complementary DNA (cDNA) template using a reaction mix containing 4 μL 5X reverse transcriptase buffer, 2 μL pDN6 50 μmol/L, 2 μL 100 mmol/L dithiothreitol, 0.2 μL 100 mmol/L dNTP mix, 4 μL 25 mmol/L MgCl2, 1 μL RNAsin 20 U/μL, and 200 U MMLV reverse transcriptase (Invitrogen). The total reaction volume was 20 μL. The mixture was incubated at 25°C for 10 minutes and subsequently at 42°C for 45 minutes. The reaction was stopped by heating at 99°C for 3 minutes. Quantitative real-time polymerase chain reaction (PCR) was performed for S100B and β-actin on an iCycler instrument from Bio-Rad Laboratories (Hercules, CA) using the Bio-Rad iCycler IQ Real-Time PCR Detection System Software (version 3.0A) to acquire and analyze data. Amplification of S100B and β-actin fragments was performed using the SYBR Green PCR master mix (Bio-Rad Laboratories).21Casabianca A. Orlandi C. Fraternale A. et al.Development of a real-time PCR assay using SYBR Green I for provirus load quantification in a murine model of AIDS.J Clin Microbiol. 2004; 42: 4361-4364Crossref PubMed Scopus (18) Google Scholar Primers sequences used were S100B forward: GTGACTTCCAGGAATTCATGGC, S100B reverse: CAGGAAAGGTTTGGCTGCTT; β-actin forward: CGACAGGATGCAGAAGGAGA, β-actin reverse: CGTCATACTCCTGCTTGCTG. Reaction conditions were 3 minutes at 95°C followed by 40 cycles at 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 20 seconds. S100B messenger RNA (mRNA) was then normalized to the respective control β-actin amount. To evaluate real-time PCR efficiencies, a 10-fold serially diluted cDNA was used for each amplicon, and the slope values given by the instrument were used in the following formula: Efficiency = [10(1/slope)] − 1. All primer sets had efficiencies of 100% (±10%). The comparative threshold cycle method against the expression level of β-actin was used for quantitation. The data are expressed as fold of increase compared with biopsy specimens cultured in medium alone after normalization to β-actin mRNA. Each experiment was performed in triplicate. Proteins extracted from the homogenized biopsy specimens (80 μg/lane) were immunoblotted with both monoclonal anti-S100B (1:1000; AbCam, Cambridge, United Kingdom) and antiinducible nitric oxide synthase (iNOS) (1:2000; Pharmingen, Milan, Italy) antibodies. After incubation with anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (1:2000; Dako, Golstrup, Denmark), immunoreactive bands were visualized by chemiluminescence (ECL+; Amersham, Milan, Italy) on x-ray film and densitometrically analyzed with a GS-700 imaging densitometer (Bio-Rad Laboratories). Enzyme-linked immunosorbent assay (ELISA) for S100B was carried out in the tissue supernatants as described by Green et al.22Green A.J. Keir G. Thompson E.J. A specific and sensitive ELISA for measuring S-100b in cerebrospinal fluid.J Immunol Methods. 1997; 205: 35-41Crossref PubMed Scopus (68) Google Scholar Briefly, 50 μL sample plus 50 μL Tris buffer were applied on a microtiter plate previously coated with monoclonal anti-S100B (AbCam) in carbonate buffer and blocked with 1% bovine serum albumin. After washing, peroxidase-conjugated anti-S100 (Dako) diluted 1:1000 was added, and incubation continued for 1 hour. The plate was washed, 0.2 mL of peroxidase substrate (Fast OPD, Sigma) was added and the plate incubated for a further 30 minutes in the dark. The absorbance was measured at 450 nm on a microtiter plate reader. S100B levels into the culture medium were determined using a standard curve of S100B and expressed as nanogram/milligram protein. In a second set of experiments, exogenous purified human S100B protein (0.005–5 μmol/L)23Adami C. Bianchi R. Pula G. et al.S100B-stimulated NO production by BV-2 microglia is independent of RAGE transducing activity but dependent on RAGE extracellular domain.Biochim Biophys Acta. 2004; 1742: 169-177Crossref PubMed Scopus (98) Google Scholar (Sigma) was added to cultured biopsy specimens deriving from controls: iNOS protein expression and relative NO production were evaluated after 24 hours. To check the presence of endotoxin, purity of S100B was assessed by the Limulus amebocyte lysate Pyrochrome assay (Cape Cod Inc., Falmouth, MA) according to the previously published method.24Lindsay G.K. Roslansky P.F. Novitsky T. Single-step, chromogenic limulus amebocyte assay for endotoxin.J Clinic Microbiol. 1989; 27: 947-951PubMed Google Scholar Lipid peroxidation, by measuring malonyldialdehyde levels according to the previously described method,25Mihara M. Uchiyama M. Determination of malonaldehyde precursor in tissue by thiobarbituric acid test.Anal Biochem. 1978; 86: 271-278Crossref PubMed Scopus (4520) Google Scholar was evaluated 6 hours after S100B (0.005–5 μmol/L) stimulus. In the same samples immunoblot analysis for phosphorilated-p38 mitogen-activated protein kinase (p-p38 MAPK) was also assessed using a monoclonal anti-p38 MAPK antibody (1:5000, Pharmingen). NO production was evaluated after S100B (5 μmol/L) stimulus in the presence or absence of 4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580 0.03–3 μmol/L) (Sigma), a p38 MAPK selective inhibitor,26Cuenda A. Rouse J. Doza Y.N. et al.SB 203580 is a specific inhibitor of a MAP kinase homologue, which is stimulated by cellular stresses and interleukin-1.FEBS Lett. 1995; 364: 229-233Abstract Full Text PDF PubMed Scopus (1998) Google Scholar or N-alpha-p-tosyl-L-lysinechlorometyl ketone (TLCK 0.01–1 μmol/L) (Sigma), a selective nuclear factor (NF)-κB transcription factor inhibitor.27Kim H. Lee H.S. Chang K.T. et al.Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-κB.J Immunol. 1995; 154: 4741-4748PubMed Google Scholar To evaluate further the specific involvement of S100B in CD and its implications in NO production, challenge experiments with gliadin were performed on cultured biopsy specimens according to reported methods.28Maiuri L. Ciacci C. Ricciardelli I. et al.Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease.Lancet. 2003; 362: 30-37Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar, 29De Ritis G. Occorsio P. Auricchio S. et al.Toxicity of wheat flour proteins and protein-derived peptides for in vitro developing intestine from rat fetus.Pediatr Res. 1979; 13: 1255-1261Crossref PubMed Scopus (81) Google Scholar In brief, duodenal biopsy specimens from controls and GFD-CD patients were incubated for 24 hours with a 1 mg/mL peptic-tryptic digest of gliadin (pt-gliadin).29De Ritis G. Occorsio P. Auricchio S. et al.Toxicity of wheat flour proteins and protein-derived peptides for in vitro developing intestine from rat fetus.Pediatr Res. 1979; 13: 1255-1261Crossref PubMed Scopus (81) Google Scholar Control experiments were performed by the addition of equal amounts of medium alone. S100B mRNA and protein expression were then measured in tissue, and the supernatant was analyzed for S100B protein release. In addition, iNOS protein expression and relative NO production were evaluated in the presence or absence of mouse monoclonal blocking antibody to S100B30Eriksen J.L. Druse M.J. Astrocyte-mediated trophic support of developing serotonin neurons: effects of ethanol, buspirone, and S100B.Brain Res Dev Brain Res. 2001; 131: 9-15Crossref PubMed Scopus (38) Google Scholar (1:100,000–1:1000 vol/vol dilution; clone SH-B4; AbCam); an IgG anti-glial fibrillary acidic protein antibody (1:1000 vol/vol dilution; AbCam) served as control. The Komolgorov-Smirnov test for normality was applied and showed the data to be normally distributed. Statistical analysis was thus performed with ANOVA and multiple comparisons with the Bonferroni test. Results were expressed as the mean ± SD of n experiments. The level of statistical significance was fixed at P < .05. In duodenal mucosal biopsy specimens from controls, immunohistochemistry demonstrated that S100B immunopositivity was most readily detectable and confined within the submucosa, with only few EGC processes extended into the mucosa (Figure 1A and 1Ai). In contrast, in CD patients, a stronger and diffuse S100B immunoreactivity was present in the submucosa, together with a marked S100B positivity in the mucosa (Figure 1B and 1Bi). Interestingly, S100B immunostaining decreased in GFD-CD patients, in whom it was mainly localized in the submucosal glial network (Figure 1C and 1Ci). Accordingly, a significant increase of both S100B mRNA (24-fold, P < .01) and protein expression (399% ± 7%, P < .01) was observed in CD patients compared with GFD-CD and controls, respectively (Figure 2A and 2B). In Figure 2C is reported that duodenal biopsy specimens of CD patients were also able to secrete significantly higher levels of S100B than GFD-CD and controls, respectively (641% ± 14%, P < .01). In basal conditions, the expression of iNOS protein and NO production were increased in the duodenal mucosa of CD patients compared with GFD-CD patients and controls (2537% ± 21% and 247% ± 6%, respectively, P < .01; Figure 3). In ex vivo experiments, we found that a 24-hour incubation of control biopsy specimens with S100B (0.005–5 μmol/L) increased in a concentration-dependent fashion both iNOS protein expression (157% ± 7% [S100B 0.005 μmol/L], P < .05; 743% ± 10% [S100B 0.05 μmol/L], 1149% ± 16% [S100B 0.5 μmol/L], and 1900% ± 12% [S100B 5 μmol/L], P < .01) (Figure 4A) and relative NO production (65.5% ± 2.5% [S100B 0.005 μmol/L], 213.8% ± 4.8% [S100B 0.05 μmol/L], P < .05; 397.4% ± 9% [S100B 0.5 μmol/L] and 749% ± 2.6% [S100B 5 μmol/L], P < .01) as compared with unstimulated biopsy specimens (Figure 4B). Also, incubation with S100B (0.005–5 μmol/L) resulted in a concentration-dependent increase in malonyldialdehyde production (385.7% ± 5.5% [S100B 0.005 μmol/L], 836.5% ± 9.6% [S100B 0.05 μmol/L], 1433% ± 16% [S100B 0.5 μmol/L], and 2693.6% ± 20% [S100B 5 μmol/L], P < .01) (Figure 5A). The lipid peroxidation increase was accompanied by a parallel and significant increase in p-p38 MAPK protein expression (161% ± 8% [S100B 0.005 μmol/L], P < .05; 677% ± 4% [S100B 0.05 μmol/L], 1561% ± 18% [S100B 0.5 μmol/L], and 3146% ± 29% [S100B 5 μmol/L], P < .01) (Figure 5B). The effect of S100B (5 μmol/L) was significantly inhibited by SB203580 (29.7% ± 4.1% [SB203580 0.03 μmol/L], 49.3% ± 3.5% [SB203580 0.3 μmol/L], and 72.6% ± 5.8% [SB203580 3 μmol/L], P < .01) and TLCK (49.3% ± 4.8% [TLCK 0.01 μmol/L], 66.0% ± 5.1% [TLCK 0.1 μmol/L], and 80.0% ± 4.4% [TLCK 1 μmol/L], P < .01), both added to cultured biopsy specimens deriving from controls, 30 minutes before S100B (5 μmol/L) challenge (Figure 6A and 6B).Figure 4(A) Western blot analysis showing the effects of exogenous S100B protein (0.005–5 μmol/L) at 24 hours in cultured duodenal biopsy specimens derived from controls. iNOS protein expression in tissue homogenates is shown in the upper panel, whereas the densitometric analysis of corresponding bands is represented in the graph. Upper panel is representative of n = 3 experiments. Each bar in the graph shows the mean ± SD of 3 experiments. *P < .05, **P < .01 vs untreated. (B) Effect of exogenous S100B protein (0.005–5 μmol/L) on NO production in cultured duodenal biopsy specimens derived from controls. NO production was determined by measuring the accumulation of nitrite in the culture medium at 24 hours. Each bar shows the mean ± SD of 6 experiments. *P < .05, **P < .01 vs untreated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5(A) Effect of 6 hours incubation of exogenous S100B protein (0.005–5 μmol/L) on malonyldialdehyde levels, as marker of lipid peroxidation, in cultured duodenal biopsy specimens derived from controls. Each bar shows the mean ± SD of 6 experiments. °°P < .01 vs untreated. (B) Western blot analysis showing the effect exogenous S100B protein (0.005–5 μmol/L) on p-p38 MAPK protein expression at 6 hours in cultured duodenal biopsy specimens derived from controls. p-p38 MAPK protein expression in tissue homogenates is shown in the upper panel, whereas the densitometric analysis of corresponding bands is represented in the graph. Upper panel is representative of n = 3 experiments. Each bar in the graph shows the mean ± SD of 3 experiments. *P < .05, **P < .01 vs untreated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Inhibitory effect of p38 MAPK inhibitor SB203580 (0.03–3 μmol/L) (A) and NF-κB inhibitor TLCK (0.01–1 μmol/L) (B) on the production of NO induced by exogenous S100B protein (5 μmol/L) in cultured duodenal biopsy specimens derived from controls. NO production was determined by measuring the accumulation of nitrite in the culture medium at 24 hours. Each bar shows the mean ± SD of 6 experiments. **P < .01 vs untreated; °°P < .01 vs S100B-treated biopsy specimens.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A significant increase of S100B mRNA was observed when GFD-CD cultured biopsy specimens were exposed to pt-gliadin (∼35-fold, P < .01), but this effect was not observed in controls (Figure 7A). Similarly, both gliadin-induced S100B protein expression (189.3% ± 7.21%, P < .01) and secretion (357.7% ± 13%, P < .01) were observed in GFD-CD patients only (Figure 7B and 7C). Moreover, challenge with pt-gliadin led to a significant increase of iNOS protein (1818.6% ± 20.2%) expression and NO production (1583% ± 20%) in biopsy specimens from GFD-CD patients. Interestingly, these effects were prevented by preincubation with the anti-S100B specific antibody (1:100,000–1:1000, vol/vol dilution), which resulted in a significant inhibition of iNOS protein expression (34.2% ± 2.3% [Ab S100B 1:100000], 60.6% ± 3.1% [Ab S100B 1:10000], and 75.7% ±3.5% [Ab S100B 1:1000] respectively, P < .01) and NO production (38.3% ± 2.9% [Ab S100B 1:100000], 52.0% ± 2.8% [Ab S100B 1:10000], and 80.7% ± 5% [Ab S100B 1:1000] respectively, P < .01) (Figure 8A–B). Nitrite production was not affected by the addition of glial fibrillary acidic protein antibody (1:1000, vol/vol dilution) before gliadin challenge in GFD-CD biopsy specimens (Figure 8B). The traditional assumption is that enteric glia serves as a supportive or nutritive element for enteric neurons. Here, we provide evidence that enteric glia is directly involved in chronic duodenal inflammation occurring in CD and that increased S100B mRNA and protein expression are accompanied with both iNOS protein expression and relative NO release. The few data compelling the role of S100B in the intestine indicate that this protein is exclusively localized in enteroglial cells,12Rhul A. Glial cells in the gut.Neurogastroenterol Motil. 2005; 17: 777-790Crossref PubMed Scopus (204) Google Scholar, 13Ferri G.L. Probert L. Cocchia D. et al.Evidence for the presence of S-100 protein in the glial component of the human enteric nervous system.Nature. 1982; 297: 409-412Crossref PubMed Scopus (193) Google Scholar, 14Neunlist M. Aubert P. Bonnaud S. et al.Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGF-β1-dependent pathway.Am J Physiol Gastrointest Liver Physiol. 2007; 292: 231-241Crossref PubMed Scopus (142) Google Scholar representing a specific marker for the identification and activation of this cell population. Several studies demonstrate that, in the brain, S100B may induce the expression of specific proinflammatory transcription factors, such as NF-κB, which promote the synthesis of NO.9Goncalves D.S. Lenz G. Karl J. et al.Extracellular S100B protein modulates ERK in astrocyte cultures.Neuroreport. 2000; 11: 807-809Crossref PubMed Scopus (64) Google Scholar, 10Lam A.G. Koppal T. Akama K.T. et al.Mechanism of glial activation by S100B: involvement of the transcription factor NF-κB.Neurobiol Aging. 2001; 22: 765-772Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 11Hu J. Ferreira A. Van Eldik L.J. S100β induces neuronal cell death through nitric oxide release from astrocytes.J Neurochem. 1997; 69: 2294-2301Crossref PubMed Scopus (321) Google Scholar We found that S100B expression was significantly increased in the duodenal mucosa of CD patients and that the degree of its expression is associated to the extent of iNOS expression. Although EGC hypertrophy has been described during gut inflammation,5Cornet A. Savidge T.C. Cabarrocas J. et al.Enterocolitis induced by autoimmune targeting of enteric glial cells: a possible mechanism in Crohn's disease?.Proc Natl Acad Sci U S A. 2001; 98: 13306-13311Crossref PubMed Scopus (270) Google Scholar our results suggest that S100B plays an active role at least in part in NO-dependent inflammation. The administration of exogenous S100B protein to noninflamed duodenal biopsy specimens deriving from controls resulted indeed in both iNOS protein expression and NO release, indicating that micromolar concentrations of this protein are able to participate to inflammatory status even in healthy duodenum. Noteworthy, in the CNS, S100B has been suggested to act through involvement of MAPK9Goncalves D.S. Lenz G. Karl J. et al.Extracellular S100B protein modulates ERK in astrocyte cultures.Neuroreport. 2000; 11: 807-809Crossref PubMed Scopus (64) Google Scholar signaling pathways and NF-κB activation.10Lam A.G. Koppal T. Akama K.T. et al.Mechanism of glial activation by S100B: involvement of the transcription factor NF-κB.Neurobiol Aging. 2001; 22: 765-772Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar This is in agreement with our previous findings demonstrating that exogenous S100B-induced iNOS protein expression and NO production occur through oxidative stress induction and relative p38 MAPK activation in rodent macrophages.31Esposito G. De Filippis D. Cirillo C. et al.Astroglial-derived S100β protein stimulates the expression of nitric oxide synthase in rodent macrophages through p38 MAP kinase activation.Life Sci. 2006; 78: 2707-2715Crossref PubMed Scopus (47) Google Scholar A growing body of evidence indicates that increased iNOS protein expression and the relative NO levels play a role in the etiophatogenesis of CD.32Daniels I. Cavill D. Murray I.A. et al.Elevated expression of iNOS mRNA and protein in celiac disease.Clin Chim Acta. 2005; 356: 134-142Crossref PubMed Scopus (32) Google Scholar In the present study, we show that, in the human duodenum, S100B mediates a significant increase in lipid peroxidation associated with a marked increase in p-p38 MAPK protein expression. It is widely known that, in different cell types, p38 MAPK phosphorilation induces iNOS protein expression and NO release via NF-κB involvement.33Kao S.J. Lei H.C. Kuo C.T. et al.Lipoteichoic acid induces nuclear factor-κB activation and nitric oxide synthase expression via phosphatidylinositol 3-kinase, Akt, and p38 MAPK in RAW 264.7 macrophages.Immunology. 2005; 115: 366-374Crossref PubMed Scopus (79) Google Scholar, 34Vega-Ostertag M. Casper K. Swerlick R. et al.Involvement of p38 MAPK in the up-regulation of tissue factor on endothelial cells by antiphospholipid antibodies.Arthritis Rheum. 2005; 52: 1545-1554Crossref PubMed Scopus (204) Google Scholar, 35Guikema B.J. Ginnan R. Singer H.A. et al.Catalase potentiates interleukin-1β-induced expression of nitric oxide synthase in rat vascular smooth muscle cells.Free Radic Biol Med. 2005; 38: 597-605Crossref PubMed Scopus (20) Google Scholar In our hands, both SB203580 and TLCK, p38 MAPK activation, and NF-κB inhibitors,26Cuenda A. Rouse J. Doza Y.N. et al.SB 203580 is a specific inhibitor of a MAP kinase homologue, which is stimulated by cellular stresses and interleukin-1.FEBS Lett. 1995; 364: 229-233Abstract Full Text PDF PubMed Scopus (1998) Google Scholar, 27Kim H. Lee H.S. Chang K.T. et al.Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-κB.J Immunol. 1995; 154: 4741-4748PubMed Google Scholar respectively, were able to inhibit S100B-dependent NO production. Although EGC are part of the very complex immunoregulatory effectors in the gut,3Ruhl A. Nasser Y. Sharkey K.A. Enteric glia.Neurogastroenterol Motil. 2004; 16: 44-49Crossref PubMed Scopus (118) Google Scholar their importance in the immune response to immunogenic stimuli is pivotal because these cells establish a strategically first defense line against foreign antigens, ie, foods, toxins, invading organisms.3Ruhl A. Nasser Y. Sharkey K.A. Enteric glia.Neurogastroenterol Motil. 2004; 16: 44-49Crossref PubMed Scopus (118) Google Scholar Similarly to their equivalent in the CNS, enteroglial cells represent a very important cell type because they are involved in immune response by acting as antigen-presenting cells.3Ruhl A. Nasser Y. Sharkey K.A. Enteric glia.Neurogastroenterol Motil. 2004; 16: 44-49Crossref PubMed Scopus (118) Google Scholar This is in agreement with a growing body of evidence indicating that EGC are a crucial component of the innate, nonspecific mucosal defense system, which regulates the expression of cytokines and several immunomodulatory molecules.36Wood J.D. Enteric neuroimmunophysiology and pathophysiology.Gastroenterology. 2004; 127: 635-657Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar On the basis of these considerations, could the observed S100B up-regulation and relative glial activation be an epiphenomenon of gut chronic inflammatory status, or is S100B a possible cytokine working as "driving force" during CD? To answer these questions, we evaluated whether pt-gliadin stimulates enteroglial-mediated expression of S100B protein and relative NO-dependent inflammation in biopsy specimens deriving from GFD-CD patients. Gliadin challenge in the biopsy specimens taken from these patients demonstrated that (1) S100B mRNA and protein expression and secretion are significantly increased by pt-gliadin exposure; and that (2) the specific anti-S100B antibody, added to the biopsy specimens before pt-gliadin challenge, significantly inhibited iNOS protein expression and NO production. A very intriguing connection between S100B and CD may be highlighted by the observation that an aberrant production of this protein, which is genetically overexpressed in the trysomic chromosome 21 in Down syndrome,37Griffin W.S. Stanley L.C. Ling C. et al.Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease.Proc Natl Acad Sci U S A. 1989; 86: 7611-7615Crossref PubMed Scopus (1710) Google Scholar may be related to the higher susceptibility of those patients to also develop CD.38Gale L. Wimalaratna H. Brotodiharjo A. et al.Down's syndrome is strongly associated with celiac disease.Gut. 1997; 40: 492-496Crossref PubMed Scopus (94) Google Scholar Although further investigations are needed to understand better the intimate connection between enteric glia and immune cells during chronic duodenal inflammation, our data suggest that EGC, via S100B up-regulation, actively participate in the NO-dependent inflammation occurring in CD.
Referência(s)