Volume 119, Issue 6, Pages 1524-1536 (December 2000) Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells  Maria T. Abreu, *, Andrew A. Palladino, *, Elizabeth T. Arnold, *, Richard S. Kwon, *, James A. McRoberts, ‡  Gastroenterology  Volume 119, Issue 6, Pages 1524-1536 (December 2000) DOI: 10.1053/gast.2000.20232 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Fas-mediated apoptosis of T84 cells. (A) Relative level of Fas surface expression on gastrointestinal epithelial cell lines. The level of Fas expression by flow cytometry is indicated by solid black lines; negative control staining is shown in gray. T84 cells are compared with Fas-sensitive IFN-γ–treated HT-29 cells and Fas-insensitive Caco-2 cells. (B) PI nuclear staining of T84 monolayers exposed to anti-Fas Ab. Basolateral but not apical exposure to anti-Fas Ab leads to T84 cell apoptosis demonstrated by nuclei with chromatin condensation (arrows) and nuclear debris. By 72 hours, basolateral cross-linking of Fas results in apoptosis, nuclear dropout (open spaces), and large nuclear outlines. Original magnification 630×. A series of images were taken through the nuclei every 0.5 μm and are shown as a projection. (C) Fas-mediated apoptosis is associated with caspase activation and cleavage of cytokeratin 18. Basolateral but not apical exposure to anti-Fas Ab leads to T84 cell apoptosis demonstrated by cytoplasmic staining for the caspase cleavage product of cytokeratin 18, a marker for epithelial cell apoptosis. Eighteen hours after basolateral cross-linking of Fas, individual green cellular outlines can be seen. By 24 hours, the fluorescence is seen in clusters of bright green debris, suggesting that individual apoptotic cells have fragmented into apoptotic bodies. Untreated monolayers gave results similar to apical exposure with anti-Fas control (not shown). Original magnification 400×. (D) Immunofluorescent staining for Fas on polarized T84 monolayers. Top panel shows the x-y (en face) plane of Fas stained T84 monolayers. Middle panel shows double-staining of nuclei (blue) and Fas (green). Lower panel shows a cross-sectional view in the x-z plane with the apical surface on the top and the basal surface on the bottom with basally situated nuclei. Predominantly lateral staining of Fas can be seen (arrows). Original magnification 630×. Gastroenterology 2000 119, 1524-1536DOI: (10.1053/gast.2000.20232) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Cross-linking of Fas causes impaired barrier function of T84 monolayers. (A) TER in T84 monolayers exposed to apical (♢) or basolateral (○) anti-Fas Ab. □, Control. The graph is an average of 6 experiments performed in duplicate; error bars indicate standard deviation. Basolateral cross-linking of Fas causes a significant decrease in TER compared with control or apically treated wells (*P < 0.0001). Average resistance of untreated T84 monolayers was 3300 ± 850 Ω · cm2, which decreased to 1460 ± 480 Ω · cm2 after basolateral cross-linking of Fas. (B) Basolateral cross-linking of Fas increases paracellular flux of [3H]mannitol in T84 monolayers. Symbols are as in A. Note apical (♢) flux is superimposed on control (□) flux curve. Mannitol flux is expressed in nmol/cm2 × 10−4. T84 monolayers were exposed apically or basolaterally to anti-Fas Ab for 24 hours, followed by measurement of [3H]mannitol flux. The graph is a representative experiment of 3 with similar findings, performed in triplicate. Flux in control or apically treated monolayers was 0.45–0.48 × 10−4 nmol · cm−2 · h−1 compared with 2.0 × 10−4 nmol · cm−2 · h−1 in monolayers exposed basolaterally to anti-Fas. (C) Apoptosis does not increase flux of dextrans of mol wt 3000 or 10,000 daltons. Dextran-FITC 3000 daltons (top panel) or dextran-FITC 10,000 daltons (bottom panel) was added to T84 monolayers that were untreated (2) or had been pretreated (♢, basolateral; ○, apical) with anti-Fas Ab for the preceding 24 hours. FITC was subsequently sampled for the next 24 hours at the indicated times. UV-irradiated T84 monolayers (▵) were used as positive controls for transcellular flux. The graph is a representative experiment of 3 with similar findings, performed in triplicate. The average flux of dextran-FITC 3000 daltons was as follows: control, 3.5 ng · cm−2 · h−1 (SD, ±2.5); apical anti-Fas, 3 ng · cm−2 · h−1 (SD, ±2); basolateral anti-Fas, 5 ng · cm−2 · h−1 (SD, ±3); and UV treatment, 80.5 ng · cm−2 · h−1 (SD, ±12.5). Flux of dextran-FITC 10,000 daltons was not detected in control or anti-Fas–treated T84 monolayers and was 14 ng · cm−2 · h−1 in UV-treated monolayers. Gastroenterology 2000 119, 1524-1536DOI: (10.1053/gast.2000.20232) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Fas-mediated apoptosis of T84 cells leads to junctional restructuring. (A) Distribution of the junctional proteins ZO-1 and desmoplakins 1 and 2 in T84 monolayers exposed apically (top) or basolaterally (bottom) to anti-Fas Ab for 72 hours. Images were analyzed with confocal microscopy at 100× magnification. In apoptotic monolayers, there are widely spaced, irregular ZO-1 and desmoplakin outlines as well as cytoplasmic staining of desmoplakin (curved arrows). Apically treated monolayers showed staining similar to control monolayers (not shown). (B) Relationship of junctional proteins to nuclei in control T84 monolayers (top) or monolayers exposed basolaterally (bottom) to anti-Fas Ab for 72 hours. Monolayers were double-stained with PI (red) and anti–ZO-1 (green) as indicated. Control monolayers contain one nucleus per junctional outline, but apoptotic monolayers have several apoptotic and intact nuclei within large junctional outlines. Control monolayers showed staining similar to apically treated monolayers (not shown). Original magnification 630×. (C) T84 cells flatten in apoptotic monolayers. Cell height of control T84 monolayers (top) was compared with cell height of T84 monolayers exposed basolaterally (bottom) to anti-Fas Ab for 72 hours. Nuclei are stained with PI (red), and tight junctions are stained with anti–ZO-1 (green). Horizontal arrows highlight the distances between tight junctions; vertical arrows indicate the height of the monolayer. x-z cuts are from 2 different experiments performed in duplicate. Control monolayers showed staining similar to apically treated monolayers (not shown). Original magnification 630×. Gastroenterology 2000 119, 1524-1536DOI: (10.1053/gast.2000.20232) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 E-cadherin staining of T84 monolayers. (A–J) Control T84 monolayers (x-y, A–C; x-z, G) and monolayers exposed to anti-Fas Ab for 24 hours (x-y, D–F; x-z, H) as indicated were stained for E-cadherin (green) and nuclei stained with PI (red). The arrows show 2 apoptotic cells that show basal staining of E-cadherin; one (white arrow) appears to be pinched off the monolayer with E-cadherin–stained cytoplasm from adjacent cells engulfing the base of the nucleus on x-z cut (H). Control monolayers showed staining similar to apically treated monolayers (not shown). (I and J) x-z cuts from separate experiments with similar findings. Vertical arrows denote cell height (I, 21.4 μm; J, 2.7 μm). Basal staining of E-cadherin was identified in 67% (±22%) of apoptotic nuclei and 20% (±14%) of intact nuclei. Original magnification 630×. (K–R) T84 monolayers treated apically with (K–N) anti-Fas or (O–R) basolaterally for 72 hours as indicated were stained for E-cadherin (red), ZO-1 (green), and nuclei stained with Hoechst (blue). A series of images was taken every 0.5 μm through the monolayers and projected to visualize the relationship between tight junctions and E-cadherin adherens junctions in apoptotic monolayers. E-cadherin–lined nuclei can be seen within a large ZO-1 outline in apoptotic monolayers. Tight junctions are located above E-cadherin junctions and thus are not colocalized and appear as separate green and red outlines. Gastroenterology 2000 119, 1524-1536DOI: (10.1053/gast.2000.20232) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Caspase inhibition protects against Fas-mediated apoptosis and maintains epithelial barrier integrity. (A) Nuclear staining of T84 monolayers exposed basolaterally to anti-Fas Ab in the presence or absence of caspase inhibitors as indicated. Nuclear staining was performed 48 hours after addition of anti-Fas Ab. Exposure to the Z-VAD-FMK caspase inhibitor reduces the number of apoptotic nuclei in T84 monolayers (right panel) compared with monolayers exposed to anti-Fas alone (left panel). Original magnification 400×. (B) TER in T84 monolayers exposed to basolateral anti-Fas Ab in the presence or absence of caspase inhibitors as indicated. The graph is an average of 3 experiments performed in duplicate; error bars indicate standard deviation. (C) Distribution of the junctional protein ZO-1 in T84 monolayers exposed basolaterally to anti-Fas Ab in the presence (right) or absence (left) of Z-VAD-FMK for 48 hours. Caspase inhibition maintains the tight junction organization in spite of Fas cross-linking. Original magnification 630×. Gastroenterology 2000 119, 1524-1536DOI: (10.1053/gast.2000.20232) Copyright © 2000 American Gastroenterological Association Terms and Conditions