No abstract

The barrier function of colonic epithelia is challenged by apoptotic loss of enterocytes. In monolayers of human colonic HT‐29/B6 cells, apoptosis induced by camptothecin was assessed by poly‐(ADP‐ribose)‐polymerase (PARP) cleavage, histone ELISA and DNA‐specific fluorochrome staining (with 4′,6′‐diamidino‐2′‐phenylindoladihydrochloride (DAPI)). Epithelial barrier function was studied in Ussing chambers by measuring transepithelial conductivity and unidirectional tracer fluxes. The ion permeability associated with single cell apoptoses was investigated with the conductance scanning technique. The spontaneous rate of apoptotic cells was 3.5 ± 0.3 % with an overall epithelial conductivity of 3.2 ± 0.1 mS cm−2. Camptothecin induced a time‐ and dose‐dependent increase of apoptosis and permeability. With 20 μg ml−1 of camptothecin for 48 h, apoptosis increased 4.1‐fold to 14.3 ± 1.5 % and the conductivity doubled to 6.4 ± 1.0 mS cm−2. While 3H‐mannitol flux increased 3.8‐fold and 3H‐lactulose flux increased 2.6‐fold, the flux of 3H‐polyethylene glycol 4000 remained unchanged. Hence, the higher permeability was limited to molecules < 4000 Da. The local epithelial conductivity was higher at the sites of apoptosis than in non‐apoptotic areas. With camptothecin the leaks associated with apoptosis became more numerous and more conductive, while in non‐apoptotic areas the conductivity remained at control level. Hence, the camptothecin‐induced increase in epithelial conductivity reflected the opening of apoptotic leaks and thus the results described, for the first time, epithelial permeability as a function of apoptosis only. The conductivity of apoptotic leaks contributed 5.5 % to the epithelial conductivity of controls and 60 % to the conductivity of monolayers treated with 20 μg ml−1 of camptothecin. Thus apoptosis increased the contribution of paracellular pathways to the overall epithelial permeability. Under control conditions the paracellular conductivity (Gpara) was smaller than the transcellular (Gtrans), but with 12 % apoptosis, Gpara exceeded Gtrans. By definition, the epithelium became ‘leaky’.

The barrier function of intestinal epithelia relies upon the continuity of the enterocyte monolayer and intact tight junctions. After incubation with tumor necrosis factor‐α TNF‐α, however, the number of strands that form the tight junctions decreases, and apoptosis is induced in intestinal epithelial cells. These morphological changes lead to a rise of transepithelial ion permeability, because the paracellular ion permeability increases and leaks associated with sites of apoptosis increase by number and magnitude. Thus apoptosis and degradation of tight junctions contribute to the increased permeability observed after exposure to TNF‐α. These mechanisms explain clinical manifestations in the inflamed intestinal wall containing cytokinesecreting macrophages‐for example, leak flux diarrhea and invasion of bacterial enterotoxins.

Colitis in interleukin-2-deficient (IL-2-/-) mice resembles ulcerative colitis in humans. We studied epithelial transport and barrier function in IL-2-/- mice and used this model to characterize mechanisms of diarrhea during intestinal inflammation. 22Na+ and 36Cl- fluxes were measured in proximal colon. Net Na+ flux was reduced from 4.0 ± 0.5 to 0.8 ± 0.5 μmol·h-1·cm-2, which was paralleled by diminished mRNA and protein expression of the Na+/H+ exchanger NHE3. Net Cl- flux was also decreased from 2.2 ± 1.6 to -2.7 ± 0.6 μmol·h-1·cm-2, indicating impaired Na+-Cl- absorption. In distal colon, aldosterone-induced electrogenic Na+ absorption was 6.1 ± 0.9 μmol·h-1·cm-2 in controls and was abolished in IL-2-/- mice. Concomitantly, mRNA expression of β- and γ-subunits of the epithelial sodium channel (ENaC) was reduced. Epithelial barrier was studied in proximal colon by impedance technique and mannitol fluxes. In contrast to ulcerative colitis, epithelial resistance was increased and mannitol fluxes were decreased in IL-2-/- mice. This was in accord with the findings of reduced ion transport as well as increased expression of tight junction proteins occludin and claudin-1, -2, -3, and -5. In conclusion, the IL-2-/- mucosa exhibits impaired electroneutral Na+-Cl- absorption and electrogenic Na+ transport due to reduced mRNA and protein expression of NHE3 and ENaC β- and γ-subunit mRNA. This represents a model of early intestinal inflammation with absorptive dysfunction due to impaired transport protein expression/function while epithelial barrier is still intact. Therefore, this model is ideal to study regulation of transporter expression independent of barrier defects.

Increased levels of tumor necrosis factor-alpha (TNF-alpha) have been found in, for example, inflammatory bowel disease (IBD) and human immunodeficiency virus (HIV) infection. To investigate a possible contribution of TNF-alpha to the pathogenesis of diarrhea in these diseases, ion transport of human distal colon was studied in the Ussing chamber in vitro. Serosal addition of TNF-alpha increased short-circuit current (Isc) of partially stripped tissues in a dose-dependent manner. Maximum Isc increase of 1.8 +/- 0.2 mumol.h-1.cm-2 was reached after 60 +/- 9 min at 200 ng/ml TNF-alpha. Bidirectional tracer flux measurements revealed that TNF-alpha induced an increase in 36 Cl serosal-to-mucosal flux, a decrease in 36Cl- mucosal-to-serosal flux, and a slight increase in K+ secretion indicated by an increased secretory 86Rb net flux. In the highly differentiated colonic epithelial cell line HT-29/B6, TNF-alpha had no effect on Isc, suggesting a mediation step located in the subepithelium. This supposition was supported by measurements on totally stripped human tissues, since removal of subepithelial layers by total stripping reduced the TNF-alpha effect by 40%. Experiments with tetrodotoxin (10(-6)M) indicated that the TNF-alpha effect was not mediated by the enteric nervous system. The specific 5-lipoxygenase blocker ICI-230487 (5 x 10(-8)M) also had no effect on TNF-alpha action. In contrast, inhibition of cyclooxygenase by indomethacin (10(-6)M inhibited the effect of TNF-alpha. Radioimmunoassay of prostaglandin E2 (PGE2) in the serosal bathing solution revealed an increase in PGE2 production/release after addition of TNF-alpha, which paralleled the Isc response. We conclude that TNF-alpha changed Cl- and K+ transport toward secretion in human colon. This effect was mediated by PGE2 produced by subepithelial cells. Thus TNF-alpha could be a mediator of diarrhea during intestinal inflammation, e.g., in IBD and HIV infection.

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).

No abstract

Cytokines are supposed to be mediators in diarrhoeal diseases. The aim of this study is to characterize the effect of tumor necrosis factor-alpha (TNFalpha) on epithelial barrier function in the colonic epithelial cell line HT-29/B6. Active ion transport and barrier function were measured as short-circuit current and transepithelial electrical resistance (Rt), respectively. In parallel, freeze-fracture electron microscopy (EM) of tight junctions (TJ) and immunofluorescence microscopy of the zonula occludens protein-1 (ZO-1) were performed. Serosal addition of TNF(alpha) (100 ng/ml) decreased Rt by 81%. This effect was dose-dependent and could be mimicked by antibodies against the p55 form of the TNF receptor. Cytotoxic effects were excluded by a negative lactate dehydrogenase (LDH) assay. Immunofluorescence localization with anti-ZO-1 antibodies revealed no evidence for disruption of the monolayer after TNFalpha treatment. In freeze-fracture EM, TJ complexity was decreased by TNFalpha, as indicated by a decrease in the number of strands from 4.7 to 3.4. The tyrosine kinase blocker genistein and the protein kinase A inhibitor H-8 reduced the effect of TNFalpha. A combination of TNFalpha with interferon-gamma acted synergistically on the epithelial barrier. In conclusion, TNFalpha impairs epithelial barrier function by altering structure and function of the tight junction, which could be of pathogenic relevance in intestinal inflammation.