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Neuro-immune regulation of macromolecular permeability in the normal human colon and in ulcerative colitis
Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Surgery in Östergötland.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background and aim: Persistent stress and life events affect the course of ulcerative colitis (UC) by largely unknown mechanisms. Regulation of epithelial permeability to antigens is crucial for the balance between inflammation and immuno-surveillance, and increased intestinal permeability has been shown in patients with ulcerative colitis. Corticotropin releasing hormone (CRH) has been implicated as an important mediator of stress-induced abnormalities in intestinal mucosal function in animal models. Further cholinergic signalling during stress

has been reported to increase bowel ion secretion in humans and uptake of HRP in rodents via activation of mast cells.

The overall aim of this thesis was to examine the role of CRH-mediated and cholinergic signalling, and their interaction with mast cells and eosinophils, in the regulation of the mucosal barrier function in the normal human colon and in UC. In vivo studies or the use of surgical specimens for such studies have major shortcomings. Therefore a method with endoscopic biopsies in Ussing chambers was established for studies of protein antigen uptake and electrophysiology in human colonic biopsies, and used in subsequent investigations.

Materials and methods: In the four studies a total of 91 healthy volunteers, 3 patients with rectal cancer, and 15 UC patients were included. Biopsies from the sigmoid colon were assessed for macromolecular permeability (Horseradish peroxidase (HRP), and 51Cr-EDTA), and electrophysiology during challenge with sodium caprate (C10), CRH or carbachol. Experiments were repeated with CRH receptor antagonists, carbachol receptor antagonists, mast cell stabilizers and nerve conductance blockers in Ussing chambers. The biopsies were examined by electron and light microscopy for endocytosis of HRP, morphological changes and receptor expression. Moreover, the human mast cell line, HMC-1; was used in studying expression of CRH receptors on mast cells.

Results: Endoscopic biopsies of human colon were viable in Ussing chambers, and the technique was shown to be a reliable tool for studies of mucosal permeability to HRP. CRH stimulates transcellular uptake of HRP in human colon via CRH receptor subtypes R1 and R2 on subepithelial mast cells. Further, carbachol acts on muscarinic receptors, located on subepithelial eosinophils. Activated muscarinic M2 and M3 receptors on increased numbers of CRHproducing eosinophils in UC, lead to activation of mast cells and increased macromolecular uptake across the colonic mucosa. This signalling cascade is previously unrecognized, and may be involved in the inflammatory process in UC.

Conclusions: In conclusion, we have demonstrated a chain of events leading to increased permeability to the protein antigen HRP in biopsies from healthy volunteers and patients with UC. The important steps begin with a cholinergic signal to muscarinic receptors on the CRH containing eosinophils. The next step includes activation of CRH receptors on mast cells leading to degranulation and increased macromolecular uptake across the epithelium. This explanatory model will have implications for understanding of the pathogenesis of UC and future treatment of the disease.

Place, publisher, year, edition, pages
Institutionen för klinisk och experimentell medicin , 2007. , 75 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1022
Keyword [en]
Colitis, ulcerative, colon, sigmoid, cytology, immunohistochemistry
National Category
Gastroenterology and Hepatology
Identifiers
URN: urn:nbn:se:liu:diva-11496ISBN: 978-91-85895-68-7 (print)OAI: oai:DiVA.org:liu-11496DiVA: diva2:17928
Public defence
2007-12-14, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2008-04-07 Created: 2008-04-07 Last updated: 2009-08-22
List of papers
1. Endoscopic biopsies in Ussing chambers evaluated for studies of macromolecular permeability in the human colon
Open this publication in new window or tab >>Endoscopic biopsies in Ussing chambers evaluated for studies of macromolecular permeability in the human colon
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2005 (English)In: Scandinavian Journal of Gastroenterology, ISSN 0036-5521, Vol. 40, no 5, 586-595 p.Article in journal (Refereed) Published
Abstract [en]

Objective Studies of mucosal permeability to protein antigens in humans are limited to in vitro techniques. The use of surgical specimens for such studies has major shortcomings. Endoscopic biopsies in Ussing chambers have been introduced as a means of studying secretion and transepithelial permeability, but have not been evaluated for studies of protein antigen uptake in human intestine.

Material and methods Standard forceps biopsies from the sigmoid colon of 24 healthy volunteers were mounted in Ussing chambers with an exposed tissue area of 1.76 mm2. 51Cr-EDTA (paracellular probe) and horseradish peroxidase (HRP; 45 kDa protein antigen) were used as permeability markers. Mucosal permeability, electrophysiology, histology and energy contents of the biopsies were studied over time. To evaluate the ability of the technique to detect permeability changes, the mucosa was modulated with capric acid, a medium-chain fatty acid, known to affect tight junctions.

Results In the Ussing chamber the mucosal biopsies were viable for 160 min with stable levels of ATP and lactate, and only minor changes in morphology. Steady-state permeability with low variability was seen for both markers during the 30-90 min period. Exposure to capric acid induced a rapid decrease in short-circuit current (Isc) and a slower reversible decrease in transepithelial resistance (TER), as well as an increased permeability to 51Cr-EDTA and HRP.

Conclusions Endoscopic biopsies of human colon are viable in Ussing chambers and are reliable tools for studies of mucosal permeability to protein antigens. The technique offers a broad potential for studies of mucosal function in the pathophysiology of human gastrointestinal diseases.

Keyword
ATP; histology; horseradish peroxidase; intestinal absorption; intestinal mucosa; lactate; short-circuit current; sodium caprate; transepithelial electrical resistance
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13152 (URN)10.1080/00365520510012235 (DOI)
Available from: 2008-04-07 Created: 2008-04-07 Last updated: 2009-06-08
2. Corticotropin releasing hormone (CRH) regulates macromolecular permeability via mast cells in normal human colonic biopsies in vitro
Open this publication in new window or tab >>Corticotropin releasing hormone (CRH) regulates macromolecular permeability via mast cells in normal human colonic biopsies in vitro
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2008 (English)In: Gut, ISSN 0017-5749, E-ISSN 1468-3288, Vol. 57, no 1, 50-58 p.Article in journal (Refereed) Published
Abstract [en]

Objective: Persistent stress and life events affect the course of ulcerativecolitis and irritable bowel syndrome by largely unknown mechanisms.Corticotropin-releasing hormone (CRH) has been implicated asan important mediator of stress-induced abnormalities in intestinalmucosal function in animal models, but to date no studies inhuman colon have been reported. The aim was to examine the effectsof CRH on mucosal barrier function in the human colon and toelucidate the mechanisms involved in CRH-induced hyper-permeability.

Design: Biopsies from 39 volunteers were assessed for macromolecularpermeability (horseradish peroxidise (HRP), 51Cr-EDTA), andelectrophysiology after CRH challenge in Ussing chambers. Thebiopsies were examined by electron and confocal microscopy forHRP and CRH receptor localisation, respectively. Moreover, CRHreceptor mRNA and protein expression were examined in the humanmast cell line, HMC-1.

Results: Mucosal permeability to HRP was increased by CRH (2.8±0.5pmol/cm2/h) compared to vehicle exposure (1.5±0.4 pmol/cm2/h),p = 0.032, whereas permeability to 51Cr-EDTA and transmucosalelectrical resistance were unchanged. The increased permeabilityto HRP was abolished by -helical CRH (9-41) (1.3±0.6pmol/cm2/h) and the mast cell stabiliser, lodoxamide (1.6±0.6pmol/cm2/h). Electron microscopy showed transcellular passageof HRP through colonocytes. CRH receptor subtypes R1 and R2were detected in the HMC-1 cell line and in lamina propria mastcells in human colon.

Conclusions: Our results suggest that CRH mediates transcellular uptake ofHRP in human colonic mucosa via CRH receptor subtypes R1 andR2 on subepithelial mast cells. CRH-induced macromolecular uptakein human colon mucosa may have implications for stress-relatedintestinal disorders.

Place, publisher, year, edition, pages
London UK: BMJ Group, 2008
Keyword
CRH receptor subtypes, barrier function, electron microscopy, human mast cell line, intestinal mucosa
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13153 (URN)10.1136/gut.2006.117549 (DOI)000251778400013 ()
Available from: 2008-04-07 Created: 2008-04-07 Last updated: 2017-12-13Bibliographically approved
3. Carbachol regulates transcellular antigen permeability in human sigmoid colon biopsies in vitro
Open this publication in new window or tab >>Carbachol regulates transcellular antigen permeability in human sigmoid colon biopsies in vitro
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Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-13154 (URN)
Available from: 2008-04-07 Created: 2008-04-07 Last updated: 2010-01-13
4. Cholinergic stimulation-induced release of CRH from eosinophils mediates increased macromolecular permeability in ulcerative colitis
Open this publication in new window or tab >>Cholinergic stimulation-induced release of CRH from eosinophils mediates increased macromolecular permeability in ulcerative colitis
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Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-13155 (URN)
Available from: 2008-04-07 Created: 2008-04-07 Last updated: 2010-01-13

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