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

ALFRED H. GITTER^*1, KERSTIN BENDFELDT^*, JÖRG-DIETER SCHULZKE and MICHAEL FROMM^*

^* Institute of Clinical Physiology,
Department of Gastroenterology, University Hospital Benjamin Franklin, Freie Universität Berlin, Berlin, Germany

¹Correspondence: Institut für Klinische Physiologie, UK Benjamin Franklin, FU Berlin, 12200 Berlin, Germany. E-mail gitter{at}medizin.fu-berlin.de

	ABSTRACT

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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 (TNF-) 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--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--induced single-cell apoptosis.

Key Words: cell death • conductance scanning • intestine • tight junction • permeability

	INTRODUCTION

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THE INTACT BARRIER of epithelia is essential for physiological homeostasis and defense against extrinsic antigens (1 , 2) . A major challenge for an uninterrupted barrier is the physiological process of cell turnover via apoptosis, but so far there is no conclusive evidence that spontaneous apoptosis causes a leak for solutes and water (3 , 4) . Morphological studies have shown that isolated cell apoptosis and cell division (5) take place without visible disruption of the epithelial cell layer; from this finding, it is suggested that the epithelial barrier is not affected (6 7 8 9 10) . Also, it is unknown whether or not induced single-cell apoptosis affects epithelial permeability (11) . Induced apoptosis can be triggered by tumor necrosis factor (TNF-), which is released in high concentration at sites of inflammation by cytokine-secreting macrophages (12) .

We therefore addressed the questions of whether a disconnection of intercellular junctions increases local epithelial permeability during spontaneous apoptosis and whether induced apoptosis takes part in TNF--induced increase in epithelial ion permeability. The measurements were performed by means of a novel conductance scanning technique (13) . It is a refinement of the voltage scanning method introduced by Frömter and Diamond (14) that had been used to detect barrier defects caused by experimental removal of single cells from Necturus gallbladder epithelium (15) . This allowed us for the first time to sense the local conductances of single apoptoses in a living epithelial tissue.

	MATERIALS AND METHODS

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Cell culture
HT-29/B6 cells represent a clone of the human colon cell line HT-29 that grows as highly differentiated polarized epithelial monolayers with properties of Cl^–- and mucus-secreting cells (16) . HT-29/B6 cells (29th passage) were seeded at a concentration of 1.6 x 10⁶ cells/cm² on Millicell-PCF support filters (PITP 01250, Millipore, Bedford, Mass.). The apical compartment of each support was routinely filled with 0.5 ml culture medium; the basolateral compartment contained 10 ml. The medium (RPMI 1640, Biochrom KG, Berlin, Germany) contained 2% stabilized L-glutamine and was enriched with 10% fetal calf serum. Culture was performed at 37°C in a 95% air, 5% CO₂ atmosphere. Confluence of the polarized monolayers was reached after 6 days and the experiments were performed on day 7.

Solutions and drugs
To provoke induced apoptosis, the serosal side of HT-29/B6 cells was incubated in culture medium containing 100 ng/ml of TNF- for 7 h. Recombinant human TNF- (10⁷ units/mg) was provided by Schering (Berlin, Germany). In the conductance scanning apparatus, mucosal and serosal surfaces of the epithelium were superfused with (concentrations in mmol/l) 113.6 NaCl, 2.4 Na₂HPO₄, 0.6 NaH₂PO₄, 21 NaHCO₃, 5.4 KCl, 1.2 CaCl₂, 1.2 MgCl₂, 10 D(+)-glucose, 10 D(+)-mannose, 2.5 L-glutamine, 0.5 ß-hydroxybutyric acid, pH 7.4, set in when the solution was gassed with carbogen (95% O₂, 5% CO₂). The temperature was kept at 37°C.

Conductance scanning
Confluent monolayers were mounted horizontally between the two half-chambers of the conductance scanning apparatus described previously (13) . The optical resolution of the setup was improved by introduction of a 40x water-immersion object lens (Zeiss, Oberkochen, Germany) and redesign of the experimental chamber to adjust it to the 2 mm working distance of the objective. The electric field, generated by sinusoidal transepithelial current (AC, 0.3 mA · cm^-2, 24 Hz) in the bath solution on the mucosal side of the epithelium, was detected with a mobile probe that was positioned 25 µm above the epithelial surface by means of a micromanipulator. The conductance probe consisted of a pair of microelectrodes (Fig. 1 ) that were connected to a differential amplifier and an AC bridge system with synchronous demodulation. Control experiments, as described previously (13) , excluded the possibility that the recordings were affected by amplitude or frequency of the current applied. Two measurements were made at each position. The current density was calculated by multiplication of the field strength measured locally and the specific resistivity of the bath solution. In nonapoptotic areas, the distribution of transepithelial current was homogeneous and the conductivity equaled the ratio of current density to transepithelial voltage. Near apoptotic rosettes, the transepithelial current was inhomogeneously elevated. Here the current associated with a single rosette was computed by numerical integration of the current density exceeding the current density of nonapoptotic areas. From rosette current and transepithelial voltage, the conductance of a single rosette was derived. All values are given as mean ± SE for 20 apoptotic rosettes in 9 control monolayers or 21 apoptoses in 11 monolayers treated with TNF-.

View larger version (24K): [in this window] [in a new window]   Figure 1. The local current density generated on the apical side of an epithelium by a transepithelial alternating current was measured with a mobile conductance probe. The latter consisted of two microelectrodes set apart vertically by 15 µm, which were connected to a differential amplifier and an AC bridge system with synchronous demodulation.

Histochemistry
Monolayers were fixed in formaldehyde and embedded in paraffin. Serial sections (3 µm) were stained with hematoxylin and eosin or dewaxed for (immuno)fluorescence localization of apoptoses. Cellular DNA was either stained with 4,6-diamidino-2-phenylindole-2-HCl (DAPI) or a TUNEL (TdT-mediated X-dUTP nick end labeling) assay (Boehringer Mannheim, Mannheim, Germany). In the latter, blunt ends of double-stranded DNA exposed by strand breaks were visualized by means of enzymatic labeling of the free 3'-OH termini with fluorescein-dUTP. Ultrathin sections for transmission electron microscopy were prepared following standard procedures.

	RESULTS

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Frequency of apoptoses
With bright-field light microscopy of HT-29/B6 monolayers, an irregular cell sheet with approximately hexagonal symmetry was found. The mean cell diameter was 6 µm. Spots where neighboring cells repositioned into a distinctive pattern of ‘rosettes’ (Fig. 2 ) were observed with a density of 3700 cm^-2. After 7 h of incubation with TNF- (100 ng/ml), the density of rosettes increased to 11,100 cm^-2 (Table 1 ). The rosette-shaped pattern of cells is also seen after staining the tight junctional complex by occludin-immunofluorescence (Fig. 2) . The same pattern can be observed after epithelial recovery from artificially induced cell loss (17) . Without experimental destruction of cells, however, the pattern may indicate apoptosis (11) .

View larger version (93K): [in this window] [in a new window]   Figure 2. Apoptosis appears as rosette pattern in the epithelial monolayer. Unstained monolayers of living HT-29/B6 cells in the experimental chamber viewed by light microscopy with bright-field illumination show clusters suggestive of a rosette, composed of radially elongated cells around a central apoptotic cell (arrow). The rosette pattern was also observed in fixed tissue stained with occludin antibody or hematoxylin and eosin (H-E). Apoptotic processes were demonstrated in the center of the rosette (white arrow) by stainings with H-E, DAPI, and TUNEL, as well as by transmission electron microscopy (TEM).

To demonstrate that in the present case the rosettes were indeed characteristic of apoptosis, cellular DNA was inspected after staining with hematoxylin and eosin, DAPI, or a TUNEL assay. Thus, apoptotic bodies were seen in the center of rosettes (Fig. 2) . Necrotic cells were not observed and lactate dehydrogenase activity, an indicator of cytolysis (18) , was not increased after TNF- treatment. Thus, like in LLC-PK1 renal epithelial cells (19) , TNF- does not produce necrosis in HT-29/B6 cells. In transmission electron micrographs, apoptotic bodies were observed in the center of rosettes and apoptotic fragments were engulfed by adjacent cells (Fig. 2) .

Epithelial ion permeability
Total transepithelial ion permeability was evaluated by measurement of transepithelial conductivity, which refers to the gross tissue area. In controls, the total epithelial conductivity (see Fig. 5 , dark bars) was 3.24 ± 0.07 mS/cm²; after TNF- treatment it increased to 14.65 ± 0.31 mS/cm². In the presence of 50 µM of the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (Z-VAD-FMK) (20) , TNF- treatment had no significant effect on epithelial conductivity.

View larger version (26K): [in this window] [in a new window]   Figure 5. Contribution of apoptoses and of nonapoptotic areas to total tissue conductivity, given per cm2 gross tissue area. Under control conditions, apoptosis contributed 5.5% to the total epithelial conductivity. With TNF-, basic epithelial conductivity of nonapoptotic areas increased 2.6-fold vs. control whereas that of apoptoses increased by a factor of 37. Hence, with TNF-, apoptosis contributed 45% to the total epithelial conductivity.

How does one decide whether tight junctional degradation or apoptosis gives rise to the increased conductivity? Conventional assessment of transepithelial ion permeability, either with tracer molecules (e.g., radioactively labeled) or by measurement of the transepithelial conductivity in conventional Ussing chambers, cannot discriminate an increased conductivity caused by single apoptotic cells from an increased conductivity caused by a general alteration in transcellular permeability (21) or degradation of the tight junctions (18) .

Nonapoptotic conductivity
The conductance scanning technique allowed for direct conductance measurements at microscopically identified epithelial structures with high spatial resolution (Fig. 1) . The spatial distribution of epithelial conductivity away from apoptotic rosettes was even, but the conductivity rose to peaks of different magnitude in the rosettes’ centers (Fig. 3 ). The basic epithelial conductivity measured in the homogeneous, nonapoptotic areas (see Fig. 5 , light bars) was 3.06 ± 0.07 mS/cm² and increased with TNF- treatment to 8.08 ± 0.05 mS/cm².

View larger version (21K): [in this window] [in a new window]   Figure 3. Current density, induced by a transepithelial stimulus and thus reflecting local conductance, at single apoptosis and in nonapoptotic area. Current density peaked in the center of apoptotic rosettes and decreased, with lateral distance from the center, toward a constant reflecting the basic epithelial conductivity in nonapoptotic areas.

Apoptotic conductivity
The conductance associated with single apoptoses was determined by measurement of the current density along a line between the rosette’s center and the area with homogeneous conductivity (Fig. 3) , integration over the area, division by the transepithelial voltage, and subtraction of basic epithelial conductivity. The histogram (Fig. 4 ) shows that under control conditions, most apoptotic rosettes exhibited conductances below 200 nS. The mean conductance of single apoptoses was 48 nS (range 0–280 nS). With TNF-, however, most apoptoses showed conductances between 200 and 600 nS, and the mean conductance of single apoptoses had increased to 597 nS (range 67–1542 nS).

View larger version (26K): [in this window] [in a new window]   Figure 4. Relative frequency of single apoptoses with different conductances. The conductance of individual apoptoses was determined under control conditions and with TNF- preincubation. The dramatic right-shift of the histogram with TNF- indicates the occurrence of apoptoses with high conductance. Note that with TNF- the number of apoptoses per area unit increased threefold (Table 1) .

The contribution of apoptoses to the total epithelial conductivity (Fig. 5 , shaded bars) was 0.18 mS/cm² or 5.5% under control conditions. With TNF-, it increased to 6.57 mS/cm² or 45% of the total epithelial conductivity (Table 1) . Thus, the TNF--induced conductance increase was based by 44% on degradation of tight junctions in nonapoptotic areas and by 56% on apoptosis-related conductivity changes, the latter indicating pronounced local barrier defects.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
So far, the lack of appropriate methods had prevented evaluation of possible local changes in epithelial permeability caused by apoptosis. The present report documents, for the first time, conductive gates formed in an epithelial monolayer by spontaneous apoptosis and, with a more pronounced effect, by TNF--induced apoptosis. Yet spontaneous apoptosis created individual leaks with a conductance up to 1000 times higher than that of large ion channels, e.g., Maxi-K⁺-channels.

With TNF-, density and conductance of apoptoses were higher than under control conditions. Thus, with induced apoptosis, the total conductivity of leaks caused by apoptosis was 37-fold higher than with spontaneous apoptosis. The higher frequency and magnitude of apoptosis-related gates in TNF--treated epithelia may be related to TNF--mediated alterations of tight junctions (18) , because cell–cell contacts can be involved in the control of apoptosis (11) .

An increase in the tight junctional ion permeability is probably responsible for the increase in the basic epithelial conductivity, because the Cl^– secretion induced by TNF- via prostaglandins is short-lived and not accompanied by significant decrease in the transepithelial resistance (22 , 23) . The caspase inhibitor Z-VAD-FMK prevented not only apoptosis, but also the rise of basic epithelial conductivity caused by TNF-. Hence, a common signaling pathway must be assumed for both effects.

Although TNF- does not disturb the morphologically visible epithelial integrity (11) , it decreases the number of strands that form the tight junctions and dramatically increases total epithelial ion conductance as well as the passive flux of paracellular markers (18 , 19 , 24 , 25) . Thus, the permeabilizing effect of TNF- has been attributed to the observed alteration of the tight junction meshwork (18 , 26) . On the other hand, TNF- also induces apoptosis (11 , 27) , particularly after induction of TNF- receptors by interferon- or blockade of protein synthesis controlled through transcription factors (28 , 29) . The present work determined quantitatively the contribution of apoptosis and nonapoptotic effects, i.e., degradation of tight junctions, to the increase of epithelial permeability induced by TNF-. It turned out that both mechanisms contributed almost equally (56 and 44%, respectively) to that increase.

The results are important for understanding the mechanisms of inflammatory processes—for instance, inflammatory bowel diseases (30 31 32) —or HIV-associated enteropathy (33) where the concentration of TNF- rises within intestinal tissues and the mucosal barrier function is impaired. The concentration of TNF- used (100 ng/ml) may reflect the local level at the site of TNF- release (34) and was only 10-fold higher than that found after distribution in the circulation of patients with ulcerative colitis (25) or Crohn’s disease (12) . Thus, our findings suggest that epithelial integrity may be compromised during TNF--mediated inflammatory processes, causing a clinical manifestation of leakiness in the intestinal wall, e.g., diarrhea (28) . Invasion of bacterial enterotoxins could then start the vicious circle proposed earlier (35 , 36) .

In conclusion, apoptosis can increase the epithelial permeability. Hence, our results supersede a major dogma of apoptotic function (4) . During regular cell renewal, spontaneous apoptosis constitutes a significant paracellular shunt, but TNF--induced apoptosis causes much larger leaks. In pathophysiological situations with increased cytokine levels, apoptosis may create distinct barrier defects.

	ACKNOWLEDGMENTS

 
We thank Dr. H. Schmitz for stimulating discussion and D. Sorgenfrei and S. Lüderitz for expert technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft and by Freie Universität Medical Faculty funds.

Received for publication December 1, 1999. Revision received March 17, 2000.

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