title: Restitution of single-cell defects in the mouse colon epithelium differs from that of cultured cells.
abstract: Integrity of colon epithelium is of crucial importance and, as small defects occur constantly, rapid repair (restitution) is essential. To investigate the mechanism of restitution, single-cell lesions were induced in mouse colonic surface epithelia by iontophoretic injection of Ca2+. Closure of the resulting defects was monitored using confocal laser scanning microscopy (CLSM), and functional sealing by electrophysiological techniques. Restitution was evaluated as the time constant tau of the exponential decrease in conductance of an induced leak and amounted to 0.28 min under control conditions. After 4 min, the leak was completely sealed. Repair was thus considerably faster than in previously investigated HT-29/B6 cells (tau=5.73 min). As in cultured cells, cytochalasin D delayed restitution in native colon epithelia (tau=0.69 min), indicating the involvement of actin in the healing process; however, no accumulation of actin surrounding the lesion was detected. Long-term incubation of epithelia with IFN-gamma alone or in combination with TNF-alpha increased tau to 0.49 and 0.59 min, respectively. In contrast to cultured cells, TNF-alpha alone did not affect restitution. A brief (<10 min) exposure to the sterile filtered supernatant of hemolytic E. coli O4 cultures did not affect the morphology of the epithelium, but delayed restitution. In CLSM studies, defects were still clearly visible 4 min after the onset of lesion induction. The supernatant of a nonhemolytic E. coli O4 mutant did not exhibit this effect. In conclusion, single-cell defects in native colon cause functional leaks that seal faster than in cell cultures. Proinflammatory cytokines and pathogenic bacteria delay restitution. This suggests a key role of very small lesions at the onset of pathogenic processes in the intestine.
authors.affiliation: Department of Clinical Physiology, Charité, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, and Jena University of Applied Sciences, Germany. dorothee.guenzel@charite.de
authors.forename: D
authors.initials: D
authors.lastname: Günzel
authors.forename: P
authors.initials: P
authors.lastname: Florian
authors.forename: J F
authors.initials: JF
authors.lastname: Richter
authors.forename: H
authors.initials: H
authors.lastname: Troeger
authors.forename: J D
authors.initials: JD
authors.lastname: Schulzke
authors.forename: M
authors.initials: M
authors.lastname: Fromm
authors.forename: A H
authors.initials: AH
authors.lastname: Gitter
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D018938:Caco-2 Cells; D002470:Cell Survival / Q000187:drug effects; D003106:Colon / Q000187:drug effects / Q000473:pathology / Q000503:physiopathology*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D004553:Electric Conductivity; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D005260:Female; D019073:HT29 Cells; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism / Q000503:physiopathology*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D008815:Mice, Inbred Strains; D018613:Microscopy, Confocal; D062793:Occludin; D012038:Regeneration / Q000187:drug effects / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D000199:Actins; D017077:Culture Media, Conditioned; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568986:Ocln protein, mouse; D014409:Tumor Necrosis Factor-alpha; D015638:Cytochalasin D; D007371:Interferon-gamma