Autocrine regulation of epithelial permeability by hypoxia: Role for polarized release of tumor necrosis factor α  Cormac T. Taylor, Andrea L. Dzus, Sean P. Colgan  Gastroenterology  Volume 114, Issue 4, Pages 657-668 (April 1998) DOI: 10.1016/S0016-5085(98)70579-7 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Epithelial exposure to hypoxia potentiates IFN-γ bioactivity. Monolayers of the human intestinal epithelial cell line T84 were grown as monolayers on permeable supports. (A and B) Confluent monolayers were exposed to hypoxia (Po2, ~20 mm Hg) in the presence of indicated concentrations of IFN-γ. (A) Baseline transepithelial resistance was measured using standard voltage clamp techniques. (B) Paracellular flux was measured using 3-kilodalton fluoresceinated dextran. Results represent means ± SEM for 7–12 individual monolayers in each condition. (C) The influence of hypoxia in combination with IFN-γ on MHC class II cell surface expression. Confluent T84 monolayers were exposed to media or media containing IFN-γ (100 U/mL) under conditions of normoxia (PO2, 147 mm Hg) or hypoxia (PO2, 20 mm Hg) for 24 hours. Cells were cooled to 4°C, and cell surface proteins were biotinylated, followed by immunoprecipitation of MHC class II as described in Materials and Methods. Blots were probed with streptavidin-peroxidase and developed by enhanced chemiluminescence. Gastroenterology 1998 114, 657-668DOI: (10.1016/S0016-5085(98)70579-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Polarized release of a hypoxia-evoked factor that potentiates IFN-γ bioactivity. Monolayers of the human intestinal epithelial cell line T84 were grown on permeable supports, and confluent monolayers were exposed to hypoxia (H; Po2, ~20 mm Hg, 24 hours) or normoxia (N). (A) Conditioned supernatants were collected and carried over to virgin monolayers in the presence of indicated concentrations of IFN-γ for 48 hours. Baseline TER values were measured using standard voltage clamp techniques and are presented as means ± SEM percent control TER. (B) Conditioned supernatants were collected from the apical (Ap) or basolateral (Ba) surface separately and carried over to virgin monolayers as above. Results represent means ± SEM for 12 individual monolayers in each condition. Gastroenterology 1998 114, 657-668DOI: (10.1016/S0016-5085(98)70579-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Epithelia release TNF-α and bear functional TNF-α receptors. Monolayers of the human intestinal epithelial cell line T84 were grown as monolayers on permeable supports, and confluent monolayers were exposed to hypoxia (▨; Po2, ~20 mm Hg, 24 hours) or normoxia (■). (A) Conditioned supernatants were collected from the apical or basolateral surface, and TNF-α was quantified from supernatants by ELISA. (B) Monolayers were exposed to media, TNF-α, or IFN-γ alone or in combination at indicated concentrations and examined for decreased TER as described above. Results represent means ± SEM for 8 individual monolayers in each condition. **Significantly different than control (P < 0.01). Gastroenterology 1998 114, 657-668DOI: (10.1016/S0016-5085(98)70579-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Influence of hypoxia on epithelial TNF-α and IFN-γ receptors. Confluent T84 monolayers were exposed to media alone or media containing IFN-γ (100 U/mL) under conditions of normoxia (N; Po2, 147 mm Hg) or hypoxia (H; Po2, 20 mm Hg) for 24 hours. Cells were cooled to 4°C, and cell surface proteins were biotinylated, followed by immunoprecipitation of (A) TNF-α receptor I (RI), (B) IFN-γ receptor (R), or (D) TNF-α receptor II (RII) as described in Materials and Methods. Blots were probed with streptavidin-peroxidase and developed by enhanced chemiluminescence. (C and E) Epithelial plasma membrane preparations from T84 cells (T84), small intestine (SI), or large intestine (LI) were immunoblotted, as indicated, for TNF-α receptor I or TNF-α receptor II. Gastroenterology 1998 114, 657-668DOI: (10.1016/S0016-5085(98)70579-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Functional up-regulation of the TNF-α receptor on intestinal epithelia. Confluent monolayers of the human intestinal epithelial cell line T84 were exposed to IFN-γ (100 U/mL, 24 hours), washed free of cytokine, and exposed to TNF-α (100 ng/mL) for indicated periods of time, and TER values were measured as described above. Results represent means ± SEM for 8 individual monolayers in each condition. ▨;, Without TNF; ■, with TNF. Gastroenterology 1998 114, 657-668DOI: (10.1016/S0016-5085(98)70579-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Thalidomide specifically diminishes epithelial TNF-α release and normalizes hypoxia-evoked potentiation of IFN-γ response. Monolayers of the human intestinal epithelial cell line T84 were grown as monolayers on permeable supports, and confluent monolayers were exposed to normoxia (NOR) or hypoxia (HYP; Po2, ~20 mm Hg, 24 hours) in the presence or absence of thalidomide (Thal). (A) Conditioned supernatants were collected from the basolateral surface, and IL-8 (▨; ng/mL) or TNF-α (■; pg/mL) was quantified from supernatants by ELISA. (B) Similarly conditioned monolayers were exposed to media or IFN-γ at indicated concentrations and examined for decreased TER as described above. Results represent means ± SEM for 9 individual monolayers in each condition. ■, Normoxia; ●, hypoxia; ▵, hypoxia plus thalidomide. CTL, control. Gastroenterology 1998 114, 657-668DOI: (10.1016/S0016-5085(98)70579-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Model of autocrine potentiation of epithelial IFN-γ response. Intestinal epithelia exposed to conditions of cellular hypoxia release, in a polarized fashion, TNF-α. This event potentiates activity of IFN-γ, a cytokine produced within the mucosal microenvironment in the baseline state, particularly during inflammation. In combination, these cytokines synergize to promote increased epithelial paracellular permeability. This model represents a novel pathway by which hypoxia and inflammation may interact in the pathogenesis of inflammatory disease. Gastroenterology 1998 114, 657-668DOI: (10.1016/S0016-5085(98)70579-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions