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First published online 25 October 2005
doi: 10.1242/jcs.02630

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Interferon- selectively increases epithelial permeability to large molecules by activating different populations of paracellular pores

¹ Gut Barrier Group, Injury Research, Salford Royal Hospitals NHS Trust and Faculty of Medicine and Human Sciences, University of Manchester, Clinical Sciences Building, Hope Hospital, Salford, M6 8HD, UK
² Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK

View larger version (17K): [in a new window]   Fig. 1. Concentration-dependent effects of IFN on PEG oligomer permeability and TER in T84 monolayers. After a 48-hour incubation with IFN, monolayers were mounted in modified Ussing chambers in HBSS. (A) Decrease in TER in response to basolateral IFN at concentrations of 0.01-10 ng/ml for 48 hours. (B) Apical to basolateral permeability profiles of PEG oligomers of increasing molecular mass (0.238-1.25 kDa) and molecular radius (3.5-7.4 Å) following a 48-hour incubation with 10 ng/ml (); 1 ng/ml () and 0.1 ng/ml () IFN or vehicle (). Permeability data is the mean of six 30-minute flux periods. Data shown are expressed as mean±s.e.m. of four monolayers in each group.

View larger version (14K): [in a new window]   Fig. 2. Correlation of IFN-induced changes in the non-restrictive pore descriptor ß with increases in permeability of FD10. (A) Changes in the values of the restrictive () and non-restrictive (ß) pore descriptors calculated from the PEG oligomer permeability profile as described in the Materials and Methods following incubation of T84 monolayers with IFN (0-100 ng/ml) for 48 hours. (B) Apparent permeability (Papp) of 10 kDa fluorescent dextran (FD10) measured under the same conditions. Data are expressed as mean±s.e.m. of n=4-6 monolayers in each group. Significant differences in Papp were observed; **P<0.01; *P<0.05 compared with control as indicated.

View larger version (14K): [in a new window]   Fig. 3. Influence of IFN on mannitol and FD10 permeability across T84 monolayers. Following incubation with IFN-(0-10 ng/ml) for 48 hours, T84 monolayers were washed with HBSS and mounted in Ussing chambers. 100 µM [14C]mannitol or 10 µM FD10 (final concentrations) were added to the apical chamber and samples removed every 30 minutes for 3 hours for analysis of probe permeation. Results are shown as fold increase in permeability of mannitol and FD10 above control. Papp values for mannitol and FD10 in control monolayers were 0.53±0.15x10–6 and 0.015±0.005x10–6 cm/second respectively. Data are expressed as mean±s.e.m. of four monolayers in each group. Significant differences in permeability were observed; **P<0.01; *P<0.05 compared with control levels as indicated.

View larger version (30K): [in a new window]   Fig. 4. Intracellular sequestration of dextran in control and IFN treated T84 monolayers. (A) Control, (B) IFN -treated (10 ng/ml; 48 hours) and (C) Ca2+-depleted T84 monolayers were incubated with 1 mg/ml Rhodamine-labelled 10 kDa dextran for 60-120 minutes as described in Materials and Methods. After fixation, monolayers were stained with the nuclear stain DAPI and analysed by confocal microscopy (x-z) images. Reconstructed confocal images in the x-z plane showing staining for Rhodamine-dextran, DAPI and merged images of the two stains are shown for each of the three groups. Dextran internalisation increased following Ca2+ depletion (areas of intense staining indicated by arrows) whereas IFN-treated monolayers showed no change compared to untreated controls. `F' denotes the position of the Transwell filter, which exhibited high levels of dextran staining in all groups. Bar, 50 µm.

View larger version (14K): [in a new window]   Fig. 5. Influence of IFN on permeability to bacterial lipopolysaccharide. (A) Control or IFN-treated T84 monolayers (10 ng/ml; 48 hours), were washed with HBSS and 0.2 mg/ml FITC-LPS added to the apical surface. The flux of FITC-LPS into the basolateral chamber was measured after 4 hours by fluorimetry and shown in µg/hour/cm2 as mean±s.e.m. of five to seven monolayers in each group. (B) Effect of inhibitors of endocytic pathways on FITC-LPS flux. Monolayers were exposed to 10 µg/ml chlorpromazine (CPZ), 10 µM LY-294002 or 3 µg/ml filipin for 15 minutes prior to addition of FITC-LPS. The IFN-mediated increase in FITC-LPS flux in the presence of inhibitors was not significantly different to that observed in the absence of inhibitors. Data are mean±s.e.m. of three to six monolayers in each group.

View larger version (47K): [in a new window]   Fig. 6. Differential Triton-X-100 solubility of tight junction proteins and the effect of IFN treatment on protein expression. (A) T84 cells were treated for 48 hours with 10 ng/ml IFN and then extracted as described in Materials and Methods. Triton-soluble (S) and Triton-insoluble (I) fractions were immunoblotted with antibodies to claudin-1, claudin-2, claudin-3 and occludin. Images are representative of at least three separate experiments. As a positive control for claudin-2, the expression of this protein in untreated Caco-2 monolayers is also shown. (B) Densitometric analyses of immunoblots of claudin-1, occludin (65 kDa) and high molecular mass (phosphorylated) occludin in the insoluble fraction of control and IFN-treated T84 monolayers. The graphs show the change in protein levels in IFN-treated monolayers as a % of control and are mean data from five separate experiments. Significant differences in protein levels were observed; *P<0.05; **P<0.01 compared with controls as indicated by paired t-test.

View larger version (19K): [in a new window]   Fig. 7. Reversibility of IFN induced alterations in paracellular permeability of PEG oligomers. Cells were treated with 10 ng/ml IFN for 48 hours. After this time IFN was removed from the medium and the cells were allowed to recover for 24 or 96 hours: , no recovery; , 24 hours; , 96 hours. A control profile (i.e. not exposed to IFN) is also shown . Data are mean±s.e.m. of four monolayers in each group.

View larger version (39K): [in a new window]   Fig. 8. Reversibility of IFN effects on claudin-1 and occludin in T84 membranes fractions. T84 monolayers were treated for 48 hours with 10ng/ml IFN followed by removal of the cytokine for a further period of 96 hours as indicated and then extracted as described in Materials and Methods. Triton-soluble (S) and Triton-insoluble (I) fractions were immunoblotted with antibodies to claudin-1 and occludin. Images are representative of three separate experiments.