Alfred H. Gitter

Leaks in the epithelial barrier caused by spontaneous and TNF-α-induced single-cell apoptosis

Current opinion assumes epithelial integrity during spontaneous apoptotic cell death. We measured, for the first time, the local conductances associated with apoptoses and show leaks of up to 280 nS (mean 48 ± 19 nS) in human intestinal epithelium. The results disprove the dogma that isolated cell apoptosis occurs without affecting the epithelial cell permeability barrier. After induction by tumor necrosis factor α (TNF‐α) the apoptotic leaks were dramatically enhanced: not only was the frequency increased by threefold, but the mean conductance also increased by 12‐fold (597±98 nS). Thus, apoptosis accounted for about half (56%) of the TNF‐α‐induced permeability increase whereas the other half was caused by degradation of tight junctions in nonapoptotic areas. Hence, spontaneous and induced apoptosis hollow out the intestinal barrier and may facilitate loss of solutes and uptake of noxious agents.—Gitter, A. H., Bendfeldt, K., Schulzke, J.‐D., Fromm, M. Leaks in the epithelial barrier caused by spontaneous and TNF‐α‐induced single‐cell apoptosis. FASEB J. 14, 1749–1753 (2000)

Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells

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.

Disrupted Barrier Function through Epithelial Cell Apoptosis

Abstract:  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‐α 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 Crohns 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‐α, interleukin (IL)‐13 is an important inductor of epithelial apoptosis in Th2 immune responses. Therapeutically,TNF‐α‐antibodies (infliximab) can restore barrier function in Crohns disease by downregulating epithelial apoptoses, while epithelial TJs are unaffected.

Permeability of human HT‐29/B6 colonic epithelium as a function of apoptosis

1 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. 2 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. 3 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. 4 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. 5 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’.

Epithelial Barrier Defects in HT-29/B6 Colonic Cell Monolayers Induced by Tumor Necrosis Factor-α

Abstract: 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.

Fast motility of isolated mammalian auditory sensory cells

Auditory sensory cells (hair cells) are responsible for sound transduction in the cochlea of the inner ear. In the presence of a longitudinal a.c. field isolated living outer hair cells showed reversible motile responses. They followed the stimulus up to at least 1 kHz. Control experiments in the presence of cytochalasin B, phalloidin and dinitrophenol excluded actomyosin as a molecular basis of the high frequency motility. The results suggest, that outer hair cells might amplify sound-induced oscillations in the inner ear and thus increase sensitivity and frequency selectivity of hearing.

Measurement of paracellular epithelial conductivity by conductance scanning.

Abstract A new method, conductance scanning, allows determination of local para- and transcellular conductivities in flat epithelia. Experiments were performed on kidney distal tubule cells, MDCK clone C11, which form monolayers on permeable supports. Above the apical surface, local voltage drops generated by a sinusoidal current clamp were recorded by means of a scanning microelectrode. Data were collected above cell centres and tight junctions. The scanning signal was always significantly higher above the tight junctions, but was uniformly distributed along the junctions. For determination of conductivities two procedures were applied. Method 1: the supraepithelial potential distribution was computed for given trans- and paracellular currents at all positions of the electrode. In a fit algorithm, the currents were varied until the calculated potential difference equalled the voltage measured. Method 2: after collecting scanning data in control Ringer’s, intercellular space width was reduced by mucosal addition of 40 mM sucrose and a second set of data was obtained at decreased paracellular, but presumably unchanged transcellular, conductivity. From these data, trans- and paracellular conductivities were calculated. Results of both methods were in excellent agreement. Confluent MDCK-C11 monolayers exhibited a transepithelial conductivity of 13 mS/cm2. The transcellular pathway contributed 2.6 mS/cm2 (20%) and the paracellular pathway 10.5 mS/cm2 (80%) to the total conductivity. Collapse of the lateral intercellular spaces decreased the paracellular conductivity to 4 mS/cm2 (60%). Confluent MDCK-C11 monolayers constitute true ”leaky” epithelia with homogeneously distributed trans- and paracellular conductivities. In conclusion, conductance scanning fills a methodical gap, which hitherto impeded the functional characterzation of tight junctions.

Supernatants of HIV-infected immune cells affect the barrier function of human HT-29/B6 intestinal epithelial cells.

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 (TNFα), interleukin-1-beta, interferon-alpha and interferon-gamma after cell–cell contact with PBMC. Supernatants of infected cells decreased transepithelial resistance (Rt), with higher effects on Rt 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. TNFα is an effector cytokine, because inhibition of TNFα 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 TNFα inducing a leak-flux which may contribute to the diarrhoea by HIV per se (HIV-enteropathy).

Active movements of the cuticular plate induce sensory hair motion in mammalian outer hair cells

It has been suggested that active motor capacities may explain the high sensitivity and sharp tuning of auditory transduction in the mammalian cochlea. Recently, slow and fast motile responses of isolated outer hair cells (OHCs) have been demonstrated. These consist of reversible length changes in the cylindrical cell body. Here we show that isolated OHCs are also capable of slow and fast movements of the cuticular plate (CP) which cause passive displacements of the stereocilia. The molecular mechanisms that underlie the slow movements appear to reside in the highly organized actomyosin cytoskeleton in the CP. Additional, fast movements of the CP were observed in OHCs which were exposed to alternating electrical fields. These rapid mechanical events followed the electrical stimulus cycle-by-cycle, and unlike the slow movements, did not depend on the actomyosin skeleton in the CP. The fast motility of the CP and the attached stereocilia suggest an additional active mechanism which may contribute to the sensitivity and the sharp tuning of the cochlea near threshold. By controlling the operating point of the reticular lamina and of the hair bundles, the slow CP and stereociliary movements may influence basilar membrane homeostasis and temporary threshold shift at high sound pressure levels.

Single-cell epithelial defects close rapidly by an actinomyosin purse string mechanism with functional tight junctions

Restitution of single‐cell defects, a frequent event in epithelia with high turnover, is poorly understood. Morphological and functional changes were recorded, using intravital time‐lapse video microscopy, confocal fluorescence microscopy, and conductance scanning techniques. After artificial single‐cell loss from an HT‐29/B6 colonic cell monolayer, the basal ends of adjacent cells extended. Concurrently, the local conductive leak associated with the defect sealed with an exponential time course (from 0.48 ± 0.05 μS 2 min post lesion to 0.17 ± 0.02 μS 8 min post lesion, n= 17). Between 3 and 10 min post lesion, a band of actin arose around the gap, which colocalized with a ring of ZO‐1 and occludin. Hence, tight junction proteins bound to the actin band facing the gap, and competent tight junctions assembled in the adjoining cell membranes. Closure and sealing were inhibited when actin polymerization was blocked by cytochalasin D, delayed following decrease of myosin‐ATPase activity by butanedione monoxime, and blocked after myosin light chain kinase inhibition by ML‐7. The Rho‐associated protein kinase inhibitor Y‐27632 did not affect restitution. After loosening of intercellular contacts in low Ca2+ Ringer solution, the time course of restitution was not significantly altered. Albeit epithelial conductivity was 12‐fold higher in low Ca2+ Ringer solution than in controls, under both conditions the repaired epithelium assumed the same conductivity as distant intact epithelium. In conclusion, epithelial restitution of single‐cell defects comprises rapid closure by an actinomyosin ‘purse‐string’ mechanism and simultaneous formation of a functional barrier from tight junction proteins also associated with the purse string.