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Defence capabilities of human intestinal epithelial cells
Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
2003 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet , 2003. , s. 75
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 862
Nyckelord [en]
Immunology, intestine, mucosa, epithelium, antimicrobial, defensins, glycocalyx, ulcerative colitis, Crohn´s disease, celiac disease
Nyckelord [sv]
Immunologi
Nationell ämneskategori
Immunologi inom det medicinska området
Forskningsämne
immunologi
Identifikatorer
URN: urn:nbn:se:umu:diva-151ISBN: 91-7305-530-1 (tryckt)OAI: oai:DiVA.org:umu-151DiVA, id: diva2:141273
Disputation
2003-12-05, E04, Byggnad 6E, Norrlands Universitetssjukhus, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2003-11-12 Skapad: 2003-11-12 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
Delarbeten
1. Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis.
Öppna denna publikation i ny flik eller fönster >>Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis.
2003 (Engelska)Ingår i: Clinical Experimental Immunology, Vol. 131, nr 1, s. 90-101Artikel i tidskrift (Refereegranskat) Published
Nationell ämneskategori
Immunologi
Identifikatorer
urn:nbn:se:umu:diva-2919 (URN)
Tillgänglig från: 2003-11-12 Skapad: 2003-11-12 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
2. Interferon-γ tempers the expression of carcinoembryonic antigen (CEA) family molecules – a role in innate colonic defence.
Öppna denna publikation i ny flik eller fönster >>Interferon-γ tempers the expression of carcinoembryonic antigen (CEA) family molecules – a role in innate colonic defence.
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2003 (Engelska)Ingår i: Scandinavian Journal of Immunology, Vol. 58, nr 6, s. 628-641Artikel i tidskrift (Refereegranskat) Published
Nationell ämneskategori
Immunologi
Identifikatorer
urn:nbn:se:umu:diva-2920 (URN)
Tillgänglig från: 2003-11-12 Skapad: 2003-11-12 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
3. Presence of bacteria and innate immunity of intestinal epithelium in childhood celiac disease
Öppna denna publikation i ny flik eller fönster >>Presence of bacteria and innate immunity of intestinal epithelium in childhood celiac disease
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2004 (Engelska)Ingår i: American Journal of Gastroenterology, ISSN 0002-9270, E-ISSN 1572-0241, Vol. 99, nr 5, s. 894-904Artikel i tidskrift (Refereegranskat) 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.

Nationell ämneskategori
Gastroenterologi
Identifikatorer
urn:nbn:se:umu:diva-21221 (URN)10.1111/j.1572-0241.2004.04157.x (DOI)15128357 (PubMedID)
Tillgänglig från: 2009-04-07 Skapad: 2009-04-07 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
4. β-Defensin-3 and -4 in intestinal epithelial cells display increased mRNA expression in ulcerative colitis
Öppna denna publikation i ny flik eller fönster >>β-Defensin-3 and -4 in intestinal epithelial cells display increased mRNA expression in ulcerative colitis
2004 (Engelska)Ingår i: Clinical and Experimental Immunology, ISSN 0009-9104, E-ISSN 1365-2249, Vol. 137, nr 2, s. 379-385Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

mRNA expression of two recently described human beta-defensins (hBD-3 and hBD-4) in epithelial cells of normal small and large intestine and the impact of chronic intestinal inflammation on their expression levels was investigated. Intestinal specimens from patients with ulcerative colitis (UC), Crohn's disease (CD) and controls with no history of inflammatory bowel disease were studied. hBD-3 and hBD-4 mRNAs were determined in freshly isolated epithelial cells by real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and by in situ hybridization. The effect of proinflammatory cytokines on hBD-3 and hBD-4 mRNA expression in colon carcinoma cells was also investigated. Purified epithelial cells of normal small and large intestine expressed both hBD-3 and hBD-4 mRNA, with higher expression levels of hBD-3 mRNA. In situ hybridization revealed higher levels of mRNA expression in the crypt- compared to the villus/luminal-compartment. Interferon (IFN)-gamma, but not tumour necrosis factor (TNF)-alpha or IL-1beta, augmented hBD-3 mRNA expression. None of these agents stimulated hBD-4 expression. Colonic epithelial cells from patients with UC displayed a significant increase in hBD-3 and hBD-4 mRNA compared to epithelial cells of controls. In contrast, small intestinal epithelial cells from CD patients did not show increased expression levels compared to the corresponding control cells. Moreover, Crohn's colitis did not show increased expression of hBD-4 mRNA, while the data are inconclusive for hBD-3 mRNA. We conclude that the chronic inflammatory reaction induced in the colon of UC patients enhances hBD-3 and hBD-4 mRNA expression in the epithelium, whereas in CD this is less evident.

Ort, förlag, år, upplaga, sidor
Oxford: Blackwell Publishing, 2004
Nyckelord
crohn's disease, cytokines, human, inflammatory bowel disease, qRT-PCR
Nationell ämneskategori
Immunologi
Identifikatorer
urn:nbn:se:umu:diva-21214 (URN)10.1111/j.1365-2249.2004.02543.x (DOI)000222711900020 ()15270856 (PubMedID)
Tillgänglig från: 2009-04-07 Skapad: 2009-04-07 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
5. Human intestinal plasma cells produce defensins
Öppna denna publikation i ny flik eller fönster >>Human intestinal plasma cells produce defensins
Visa övriga...
Manuskript (Övrigt vetenskapligt)
Identifikatorer
urn:nbn:se:umu:diva-2923 (URN)
Tillgänglig från: 2003-11-12 Skapad: 2003-11-12 Senast uppdaterad: 2010-01-13Bibliografiskt granskad

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