RESUMEN
Listeria monocytogenes is a food-borne pathogen, which is able to induce diarrhea when residing in the intestine. We studied the effect of listeriolysin O (LLO), an extracellular virulence factor of L. monocytogenes, on intestinal transport and barrier function in monolayers of HT-29/B6 human colon cells using the Ussing technique to understand the pathomechanisms involved. Mucosal addition of LLO, but not a LLO mutant, induced a dose- and pH-dependent increase in short-circuit current (I(SC)). Sodium and chloride tracer flux and DIDS sensitivity studies revealed that I(SC) was mainly due to electrogenic chloride secretion. Barrier function was impaired by LLO, as assessed by transepithelial resistance (R(t)) and mannitol flux measurements. Intracellular signal transduction occurred through Ca(2+) release from intracellular stores and PKC activation. In conclusion, listeriolysin induces chloride secretion and perturbs epithelial barrier function, thus potentially contributing to Listeria-induced diarrhea.
Asunto(s)
Toxinas Bacterianas/farmacología , Cloruros/metabolismo , Células Epiteliales/metabolismo , Proteínas de Choque Térmico/farmacología , Proteínas Hemolisinas/farmacología , Mucosa Intestinal/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Toxinas Bacterianas/genética , Transporte Biológico/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quelantes/farmacología , Colforsina/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Impedancia Eléctrica , Fenómenos Electrofisiológicos/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células HT29 , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Humanos , L-Lactato Deshidrogenasa/metabolismo , Manitol/metabolismo , Permeabilidad/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Recombinantes/farmacología , Sodio/metabolismo , Tapsigargina/farmacología , Teofilina/farmacologíaRESUMEN
Extraintestinal pathogenic Escherichia coli (ExPEC) are usually harmless colonizer of the intestinal microflora. However, they are capable to translocate and cause life-threatening disease. Translocation of ExPEC isolates was quantified in colonic monolayers. Transepithelial resistance (R(t)) was monitored and local changes in conductivity analysed with conductance scanning. Confocal microscopy visualized the translocation route. Corroboratory experiments were performed on native rat colon. One translocating strain E. coli O4 was identified. This translocation process was associated with an R(t) decrease (36 +/- 1% of initial resistance) beginning only 2 h after inoculation. The sites of translocation were small defects in epithelial integrity (focal leaks) exhibiting highly increased local ion permeability. Translocation was enhanced by preincubation of monolayers with tumour necrosis factor-alpha or interleukin-13. Mutant strains lacking alpha-haemolysin lost the ability to induce focal leaks, while this effect could be restored by re-introducing the haemolysin determinant. Filtrate of a laboratory strain carrying the alpha-haemolysin operon was sufficient for focal leak induction. In native rat colon, E. coli O4 decreased R(t) and immunohistology demonstrated focal leaks resembling those in cell monolayers. E. coli alpha-haemolysin is able to induce focal leaks in colonic cell cultures as well as in native colon. This process represents a novel route of bacterial translocation facilitated by pro-inflammatory cytokines.
Asunto(s)
Colon/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Proteínas Hemolisinas/metabolismo , Mucosa Intestinal/metabolismo , Animales , Colon/microbiología , Interleucina-13/farmacología , Mucosa Intestinal/microbiología , Masculino , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Epithelial barrier function is determined by trans- and paracellular permeabilities, the latter of which is mainly influenced by tight junctions (TJs) and apoptotic leaks within the epithelium. The present article aims to present experimental evidence for a functional role of epithelial apoptoses by means of cell culture models as well as in tissues from patients with inflammatory bowel disease. It is shown that epithelial apoptoses are sites of elevated conductance within the intestinal epithelium and that proinflammatory cytokines like TNF-alpha upregulate both the apoptotic rate and single apoptotic conductivity, making cytokine-induced apoptosis functionally far more relevant than is spontaneous apoptosis. In ulcerative colitis and Crohn's disease (CD), but not in collagenous colitis, apoptotic rates are increased to about 5%, in mild-to-moderately inflamed colon specimens, where as the control apoptotic rate is about 2%. Thus, epithelial apoptoses lead to a loss of ions and water into the intestinal lumen, causing leak flux diarrhea and enabling small antigens of <4,000 Da in the intestinal lumen to enter the intestinal mucosa, thereby perpetuating inflammatory responses. In addition to TNF-alpha, interleukin (IL)-13 is an important inductor of epithelial apoptosis in Th2 immune responses. Therapeutically,TNF-alpha-antibodies (infliximab) can restore barrier function in Crohn's disease by downregulating epithelial apoptoses, while epithelial TJs are unaffected.
Asunto(s)
Apoptosis/fisiología , Colitis Ulcerosa/fisiopatología , Mucosa Intestinal/fisiopatología , Línea Celular , Colitis Ulcerosa/patología , Colon/fisiopatología , Conductividad Eléctrica , Impedancia Eléctrica , Humanos , Mucosa Intestinal/patología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.
Asunto(s)
Células Epiteliales/metabolismo , Proteínas de la Membrana/metabolismo , Uniones Estrechas/fisiología , Western Blotting , Células CACO-2 , Comunicación Celular , Permeabilidad de la Membrana Celular , Claudina-5 , Impedancia Eléctrica , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Células HT29 , Humanos , Manitol/metabolismo , Proteínas de la Membrana/genética , Microscopía Confocal , Ocludina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by a Th2 immune response with inflammation and epithelial barrier dysfunction. So far, Th2 cytokines have not been shown to directly influence epithelial barrier function. METHODS: Lamina propria mononuclear cells (LPMCs) were stimulated and interleukin (IL)-13 was measured by enzyme-linked immunosorbent assay. Functional IL-13 and IL-4 effects were studied on HT-29/B6 colonic epithelial cells in Ussing chambers and by conductance scanning. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. IL-13/IL-4 receptors were analyzed by reverse-transcription polymerase chain reaction and immunofluorescence. Western blotting combined with immunofluorescence was used to detect tight junction proteins. Furthermore, restitution velocity was measured. Finally, mucosal biopsy specimens from patients with UC were compared with cultured cells for these features. RESULTS: LPMCs from patients with UC produced large amounts of IL-13 (985 +/- 73 pg/mL), much more than from controls or patients with Crohn's disease. IL-13Ralpha1 and IL-4Ralpha receptors were present in HT-29/B6 cells and colonic epithelial cells of control patients and patients with UC. IL-13 had a dose-dependent effect on transepithelial resistance of HT-29/B6 monolayers (reduction to 60% +/- 4%), whereas IL-4 had no effect. This was due to an increased number of apoptotic cells (5.6-fold +/- 0.9-fold) and an increased expression of the pore-forming tight junction protein claudin-2 to 295% +/- 37%, both of which contributed equally. Finally, epithelial restitution velocity decreased from 15.1 +/- 0.6 to 10.6 +/- 0.5 microm/h after treatment with IL-13. Parallel changes were observed in human samples, with an increase in claudin-2 expression to 956% +/- 252%. CONCLUSIONS: IL-13 was identified as an important effector cytokine in UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity.
Asunto(s)
Apoptosis/inmunología , Colitis Ulcerosa/inmunología , Citocinas/metabolismo , Interleucina-13/metabolismo , Uniones Estrechas/inmunología , Adulto , Apoptosis/fisiología , Biomarcadores/análisis , Biopsia con Aguja , Western Blotting , Proliferación Celular , Células Cultivadas , Colitis Ulcerosa/patología , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Citocinas/análisis , Células Epiteliales/citología , Femenino , Humanos , Inmunohistoquímica , Interleucina-13/análisis , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Probabilidad , Pronóstico , Factores de Riesgo , Muestreo , Sensibilidad y Especificidad , Células Th2/inmunologíaRESUMEN
BACKGROUND & AIMS: Small epithelial wounds heal by purse-string contraction of an actomyosin ring that is regulated by myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK). These studies aimed to define the roles of these kinases in purse-string wound closure. METHODS: Oligocellular and single-cell wounds were created in intestinal epithelial monolayers. Fluorescence imaging and electrophysiologic data were collected during wound closure. Human biopsies were studied immunohistochemically. RESULTS: Live-cell imaging of enhanced green fluorescent protein-beta-actin defined rapid actin ring assembly within 2 minutes after wounding. This progressed to a circumferential ring within 8 minutes that subsequently contracted and closed the wound. We therefore divided this process into 2 phases: ring assembly and wound contraction. Activated rho and ROCK localized to the wound edge during ring assembly. Consistent with a primary role in the assembly phase, ROCK inhibition prevented actin ring assembly and wound closure. ROCK inhibition after ring assembly was complete had no effect. Recruitment and activation of MLCK occurred after ring assembly was complete and coincided with ring contraction. MLCK inhibition slowed and then stopped contraction but did not prevent ring assembly. MLCK inhibition also delayed barrier function recovery. Studies of human colonic biopsy specimens suggest that purse-string wound closure also occurs in vivo, because MLC phosphorylation was enhanced surrounding oligocellular wounds. CONCLUSIONS: These results suggest complementary roles for these kinases in purse-string closure of experimental and in vivo oligocellular epithelial wounds; rho and ROCK are critical for actin ring assembly, while the activity of MLCK drives contraction.
Asunto(s)
Mucosa Intestinal/fisiopatología , Quinasa de Cadena Ligera de Miosina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Cicatrización de Heridas , Citoesqueleto de Actina , Actinas/metabolismo , Células CACO-2 , Colon/metabolismo , Colon/patología , Colon/fisiopatología , Sistemas de Computación , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Enfermedades Inflamatorias del Intestino/fisiopatología , Mucosa Intestinal/enzimología , Mucosa Intestinal/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Cadenas Ligeras de Miosina/metabolismo , Permeabilidad , Fosforilación , Factores de Tiempo , Distribución Tisular , Quinasas Asociadas a rhoRESUMEN
Tight junctions seal the paracellular pathway of epithelia but, in leaky tissues, also exhibit specific permeability. In order to characterize the contribution of claudin-2 to barrier and permeability properties of the tight junction in detail, we studied two strains of Madin-Darby canine kidney cells (MDCK-C7 and MDCK-C11) with different tight junctional permeabilities. Monolayers of C7 cells exhibited a high transepithelial resistance (>1 kOhms cm(2)), compared with C11 cells (<100 Ohms cm(2)). Genuine expression of claudin-1 and claudin-2, but not of occludin or claudin-3, was reciprocal to transepithelial resistance. However, confocal microscopy revealed a marked subjunctional localization of claudin-1 in C11 cells, indicating that claudin-1 is not functionally related to the low tight junctional resistance of C11 cells. Strain MDCK-C7, which endogenously does not express junctional claudin-2, was transfected with claudin-2 cDNA. In transfected cells, but not in vector controls, the protein was detected in colocalization with junctional occludin by means of immunohistochemical analyses. Overexpression of claudin-2 in the originally tight epithelium with claudin-2 cDNA resulted in a 5.6-fold higher paracellular conductivity and relative ion permeabilities of Na(+) identical with 1, K(+)=1.02, NMDG(+)=0.79, choline(+)=0.71, Cl(-)=0.12, Br(-)=0.10 (vector control, 1:1.04:0.95:0.94:0.85:0.83). By contrast, fluxes of (radioactively labeled) mannitol and lactulose and (fluorescence labeled) 4 kDa dextran were not changed. Hence, with regular Ringer's, Na(+) conductivity was 0.2 mS cm(-2) in vector controls and 1.7 mS cm(-2) in claudin-2-transfected cells, while Cl(-) conductivity was 0.2 mS cm(-2) in both cells. Thus, presence of junctional claudin-2 causes the formation of cation-selective channels sufficient to transform a 'tight' tight junction into a leaky one.
Asunto(s)
Canales Iónicos/biosíntesis , Proteínas de la Membrana/genética , Animales , Secuencia de Bases , Cationes , Línea Celular , Claudinas , Cartilla de ADN , Perros , Técnica del Anticuerpo Fluorescente , Ratones , TransfecciónRESUMEN
OBJECTIVES: Characterization of the diarrhoea-inducing effect of altered cytokine production in HIV infection. METHODS: Monocyte-derived macrophages (MDM) were infected with macrophagetropic (SF162) and lymphocytotropic (IIIB) HIV-1 strains and cocultured with autologous peripheral blood mononuclear cells (PBMC). After 24 h the supernatants were collected and tested for their immunoreactive levels of cytokines by enzyme-linked immunosorbent assay. The effects of the supernatants and the respective recombinant human cytokines on barrier function of HT-29/B6 cells were determined. RESULTS: Infection of MDM with HIV-1 SF162 or IIIB led to increased production of tumour necrosis factor-alpha (TNFalpha), interleukin-1-beta, interferon-alpha and interferon-gamma after cell-cell contact with PBMC. Supernatants of infected cells decreased transepithelial resistance (R(t)), with higher effects on R(t) in HIV IIIB infection, which was due to higher cytokine concentrations. The effect was not due to cytotoxicity (negative LDH assay) or epithelial monolayer disruption [zonula occludens protein-1 (ZO-1) immunofluorescence staining]. The effect of HIV-1 IIIB coculture supernatants could be mimicked by the respective recombinant human cytokines. TNFalpha is an effector cytokine, because inhibition of TNFalpha by its soluble receptor decreased the effect of the supernatants on transepithelial resistance. Conductance scanning indicated the cytokine-induced barrier defect to be due to both, induction of epithelial apoptoses and tight junction alterations. CONCLUSIONS: Cell-cell interaction of HIV-infected macrophages with PBMC leads to a release of cytokines sufficient to alter intestinal epithelial barrier function. The main effect was mediated by TNFalpha inducing a leak-flux which may contribute to the diarrhoea by HIV per se (HIV-enteropathy).