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Presence of bacteria and innate immunity of intestinal epithelium in childhood celiac disease
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Paediatrics.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
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2004 (English)In: American Journal of Gastroenterology, ISSN 0002-9270, E-ISSN 1572-0241, Vol. 99, no 5, 894-904 p.Article in journal (Refereed) Published
Abstract [en]

OBJECTIVES: Exposure to gliadin and related prolamins and appropriate HLA-DQ haplotype are necessary but not sufficient for contracting celiac disease (CD). Aberrant innate immune reactions could be contributing risk factors. Therefore, jejunal biopsies were screened for bacteria and the innate immune status of the epithelium investigated.

METHODS: Children with untreated, treated, challenged CD, and controls were analyzed. Bacteria were identified by scanning electron microscopy. Glycocalyx composition and mucin and antimicrobial peptide production were studied by quantitative RT-PCR, antibody and lectin immunohistochemistry.

RESULTS: Rod-shaped bacteria were frequently associated with the mucosa of CD patients, with both active and inactive disease, but not with controls. The lectin Ulex europaeus agglutinin I (UEAI) stained goblet cells in the mucosa of all CD patients but not of controls. The lectin peanut agglutinin (PNA) stained glycocalyx of controls but not of CD patients. mRNA levels of mucin-2 (MUC2), alpha-defensins HD-5 and HD-6, and lysozyme were significantly increased in active CD and returned to normal in treated CD. Their expression levels correlated to the interferon-gamma mRNA levels in intraepithelial lymphocytes. MUC2, HD-5, and lysozyme proteins were seen in absorptive epithelial cells. beta-defensins hBD-1 and hBD-2, carcinoembryonic antigen (CEA), CEA cell adhesion molecule-1a (CEACAM1a), and MUC3 were not affected.

CONCLUSIONS: Unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients. These glycosylation differences could facilitate bacterial adhesion. Ectopic production of MUC2, HD-5, and lysozyme in active CD is compatible with goblet and Paneth cell metaplasia induced by high interferon-gamma production by intraepithelial lymphocytes.

Place, publisher, year, edition, pages
2004. Vol. 99, no 5, 894-904 p.
National Category
Gastroenterology and Hepatology
Identifiers
URN: urn:nbn:se:umu:diva-21221DOI: 10.1111/j.1572-0241.2004.04157.xPubMedID: 15128357OAI: oai:DiVA.org:umu-21221DiVA: diva2:211049
Available from: 2009-04-07 Created: 2009-04-07 Last updated: 2015-03-18Bibliographically approved
In thesis
1. Innate and adaptive immunity in childhood celiac disease
Open this publication in new window or tab >>Innate and adaptive immunity in childhood celiac disease
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Celiac disease (CD) is an inflammatory small-bowel enteropathy caused by a permanent intolerance to wheat gluten and related proteins in rye and barley. Even though the disease originate from the small intestine the clinical symptoms varies in affected individuals and are often different in small children compared to adolescents and adults. Susceptibility to develop the disease is strongly associated with certain genetic factors i.e. HLADQ2/DQ8 but it is undoubtedly that additional inherited and environmental factors are involved. As specific T lymphocyte reactions are central in the pathogenesis of CD, six key cytokine messenger RNA levels in intestinal intraepithelial and lamina propria T lymphocytes (IEL, LPL), retrieved from small intestinal biopsies, were determined by using quantitative real-time reverse transcription polymerase chain reaction (RTPCR). Levels of cytokines, small secreted proteins which mediate and regulate immunity, in children with active disease were compared with that of treated children and controls. Interferon (IFN)-γ and interleukin (IL)-10 were also determined at the protein level by immunohistochemistry. Active celiac disease was characterized by distortions in cytokine expression, with highly significant increases of IFN-γ and IL-10 but no concomitant increases in tumor necrosis factor α (TNF-α), transforming growth factor β1 (TGFβ1), or IL-2 and no induction of IL-4. A marked shift of IFN-γ and IL-10 production from LPLs to IELs was characteristic of active celiac disease, and as many as one fourth of the IELs expressed IFN-γ. IELs in treated, symptom-free celiac patients still had increased IFN-γ levels compared with controls. In CD, gluten intake seems to cause an overreaction in IELs, with uncontrolled production of IFN-γ and IL-10 which may cause both recruitment of more IELs and a leaky epithelium, leading to a vicious circle with amplified immune activity and establishment of the intestinal lesion. In order to determine different IEL subsets contribution of the produced cytokines, γδIELs, CD4+αβIELs, and CD8+αβIELs as well as CD94+CD8+αβIELs and CD94CD8+αβIELs of children with active CD and children with no food-intolerance were analyzed for cytokine mRNA expression levels by RT-PCR. In active CD, CD8+αβIELs had the highest expression levels of IFN-γ- and IL-10 mRNA and constituted the cellular source for almost all IFN-γ and a large fraction of the IL-10. Expression levels of these two cytokines correlated and were higher in CD94-CD8+αβIELs than CD94+CD8+αβIELs CD4+αβIELs had the highest expression levels of TNF-α and despite the small number of this cell subset they contributed with half of the small amounts of this cytokine. Interestingly, TNF-α levels correlated with IL-10 in CD4+αβIELs. γδIELs had the lowest expression levels of IFN-γ, TNF-α, IL-10, and TGF-β1. Essentially no IL-2 mRNA was detected in the three IEL subpopulations. “Classical” CD8+CD94-αβT cells in the epithelial compartment are responsible for most of the excessive production of proinflammatory IFN-γ. The question whether an impaired extrathymic T cell maturation and/or capacity for secondary T cell receptor (TCR) gene recombination in iIELs is a contributing factor to CD was addressed. Expression levels of recombination activating gene-1 (RAG1) and the pre T α-chain (preTα) mRNAs were determined in IEL T cell lineage subsets of children with CD and controls. In controls, RAG1 was expressed in both mature (TCRγδ+ and TCRαβ+) and immature (CD2+CD7+TCR-) IELs while preTα was expressed preferentially in immature IELs. The RAG1 splice form selectively expressed outside thymus (RAG1 1A/2) as well as preTα were significantly decreased in CD patients both in active and inactive disease suggesting a deteriorated capacity of de novo TCR gene rearrangement in local T cell development and / or of secondary TCR gene rearrangement during editing or antigen-driven revision. This may lead to an imbalance between thymus- and gut derived T lymphocytes in the intestinal mucosa with consequent inefficient regulation of T cell responses against food antigens. Innate or nonspecific immunity is the first line, immediate defense against pathogens mediated by the epithelial cells in the intestine (IECs). As certain adaptive immune reaction in CD mimics that of intestinal infections, aberrant innate immune reaction could be a contributing factor to CD. Therefore jejunal biopsies were screened for bacteria and the innate immune status of the epithelium was investigated. Bacteria were freqently (40%) associated with the mucosa of children with active but also treated disease (20%) compared to controls (2%). Lack of antimicrobial factors such as mucins, proteins forming protective biofilm on the IECs, defensins and lysozym, peptides and enzymes with antibacterial effects, could not explain the presence of bacteria. If anything, mucin-2 (MUC2), α-defensins, HD-5, HD-6, and lysozyme mRNA levels were increased in epithelial cells in active CD, returning to normal levels in treated CD. Their expression levels correlated to the IFN-γ mRNA levels in IELs. Analysis of beta defensins, hBD-1 and hBD-2 as well as carcinoembryonic antigen (CEA) cell adhesion molecule 1a (CECAM1a), glycoproteins in the glycocalyx with ability to bind micro organisms, were not affected by the disease. Lectin staining by histochemistry revealed that goblet cells were stained by UEA1 in CD both active and treated but not in controls. The opposite pattern was seen for the lectin PNA where staining was seen in controls in the glycocalyx layer but not in CD. Thus altered glycocalyx/mucous layer may promote bacterial adhesion in CD.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2006. 69 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1054
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-874 (URN)91-7264-162-2 (ISBN)
Public defence
2006-10-06, Astrid Fagreussalen, 6A, NUS, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2006-09-25 Created: 2006-09-25 Last updated: 2013-03-25Bibliographically approved
2. Defence capabilities of human intestinal epithelial cells
Open this publication in new window or tab >>Defence capabilities of human intestinal epithelial cells
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The epithelial cells lining the intestinal mucosa separate the underlying tissue from components of the intestinal lumen. Innate immunity mediated by intestinal epithelial cells (IECs) provides rapid protective functions against microorganisms. Innate immunity also participates in orchestrating adaptive immunity. Key components in innate defence are defensins.

To study the production of defensins and how it is affected by intestinal inflammation IECs were isolated from the small and large intestines of patients suffering from ulcerative colitis (UC), Crohn´s disease (MbC), celiac disease (CD), and from controls, and analyzed by quantitative RT-PCR (qRT-PCR) and immunoflow cytometry. Defensin expressing cells were also studied by in situ hybridization and immunohistochemistry.

Normally, only small intestinal Paneth cells express human α-defensin 5 (HD-5) and HD-6. In UC colon IECs, HD-5, HD-6, and lysozyme mRNAs were expressed at high levels. In Crohn´s colitis colon the levels of HD-5 and lysozyme mRNAs were also increased although not to the same extent as in UC. No increase was detected in MbC with ileal localization. Metaplastic Paneth cell differentiation in UC colon was primarily responsible for the expression of the antimicrobial components. Human β-defensin 1 (hBD-1) mRNA was more abundant in large than in small intestine of controls, and remained unchanged in UC and MbC. hBD-2 mRNA was barely detectable in normal intestine and was induced in UC IECs but not in MbC IECs. mRNAs for the recently discovered hBD-3 and hBD-4, were detected in IECs from both small and large intestine. Both hBD-3 and hBD-4 mRNA were significantly increased in IECs of UC patients but not of MbC patients. Bacteria and IL-1β induced hBD-2 but not hBD-1 mRNA in colon carcinoma cell lines. IFN-γ, but not TNF-α or IL-1β, augmented hBD-3 expression in these cells, while none of the agents induced hBD-4. High antimicrobial activity of IECs in UC may be a consequence of changes in the epithelial lining, which permit the adherence of microorganisms.

Unexpectedly, in situ hybridization revealed expression of hBD-3 and hBD-4 mRNAs by numerous lamina propria cells in colonic tissue from UC patients. These cells were identified as plasma cells (CD138+). hBD-3 and hBD-4 mRNAs were also demonstrated in the plasmacytoma cell line U266. This is the first demonstration of defensins in plasma cells.

The four prominent constituents of the intestinal glycocalyx, carcinoembryonic antigen (CEA), CEA cell adhesion molecule 1 (CEACAM1), CEACAM6 and CEACAM7 all seem to play a critical role in innate defence of the intestinal mucosa by trapping and expelling microorganisms at the epithelial surface. The inducibility of these molecules in colonic epithelial cell lines was analyzed by qRT-PCR, immunoflow cytometry, and immunoelectron microscopy. IFN-g but not bacteria, LPS, TNF-α, or IL-1β modified the expression of CEA, CEACAM1 and CEACAM6. None of these agents modified CEACAM7 expression. IFN-γ was shown to have two effects: a direct effect on CEACAM1 transcription, and promotion of cell differentiation resulting in increased CEA and CEACAM6 and decreased CEACAM7 expression.

Scanning electron microscopy of jejunal biopsies from children with CD revealed the presence of rod shaped bacteria in ~40% of patients with active CD, but only in 2% of controls. 19% of treated CD patients still had adhering bacteria. Presence of bacteria is not due to lack of antimicrobial factors. In fact, HD-5, HD-6, and lysozyme mRNA levels were significantly increased in IECs of patients with active CD. hBD-1 and hBD-2 were unchanged. Lack of induction of hBD-2 may reflect disturbed signalling in IECs of CD patients. Analysis of CEA and CEACAM1 mRNA/protein expression showed no differences between CD patients and controls. Analysis of the mucins MUC2 and MUC3 revealed significantly increased MUC2 levels in active disease and unchanged MUC3. Immunohistochemistry demonstrated goblet cell metaplasia as well as staining of the apical portion of absorptive cells. Glycosylation status of proteins was studied by lectin histochemistry. Goblet cells in the mucosa of CD patients were stained by the lectin UEAI. This was not seen in controls. The lectin PNA stained the glycocalyx of controls but not that of CD patients. Thus, unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients and may allow bacterial binding.

We conclude that the intestinal epithelium is heavily involved in the innate defence of the mucosa and that its reactive pattern is affected by intestinal inflammation.

Keywords: human intestinal mucosa; epithelial cells; innate immunity; defensin; ulcerative colitis; Crohn´s disease; celiac disease; glycoαcalyx; mucin

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2003. 75 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 862
Keyword
Immunology, intestine, mucosa, epithelium, antimicrobial, defensins, glycocalyx, ulcerative colitis, Crohn´s disease, celiac disease, Immunologi
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-151 (URN)91-7305-530-1 (ISBN)
Public defence
2003-12-05, E04, Byggnad 6E, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2003-11-12 Created: 2003-11-12 Last updated: 2017-01-31Bibliographically approved

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Fahlgren, AnnaHörstedt, PerHammarström, StenHernell, OlleHammarström, Marie-Louise

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Forsberg, GöteFahlgren, AnnaHörstedt, PerHammarström, StenHernell, OlleHammarström, Marie-Louise
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PaediatricsImmunology/ImmunchemistryDepartment of Plant PhysiologyUmeå Plant Science Centre (UPSC)
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American Journal of Gastroenterology
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