3780

Functional Profiling Of Intestinal Tight Junctions In Vitro Highlights Mechanistic Differences Between Tight Junction Modulators. Christopher J. Watson, Malcolm Rowland, Geoff Warhurst, Univ of Manchester, Manchester United Kingdom

Background: Intestinal tight junctions (T J) form a crucial protective barrier that allows paracel- lular diffusion of ions and small solutes but excludes potentially toxic macromolecules and microorganisms. The molecular changes that accompany TJ modulation by physiological, pathological and pharmacological stimuli are increasingly well characterized. However, our understanding of the functional characteristics of the parace~ular pathway and how these are altered by modulation is limited. This is due primarily to the limitations of conventional paracellular probes. Methods: To address this we have developed an in vitro profiling technique that measures simultaneously the permeabilities of 25 polyethylene glycol oligomers of increas- ing hydrodynamic radius (0.35-0.74nm) by liquid chromatography-mess spectroscopy (LC- MS). These data combined with a mathematical sieving model provide quantitative descriptors of the paracellular route including pore size and abundance. Results: In 184 monolayers, the PEG profile conformed to a biphasic process involving a population of restrictive pores (~x) of radius O.43nm and a smaller number of pores (,8) that were non-restrictive for these PEG oligomers. PEG profiling revealed significant differences between the effects of the TJ modulators EGTA (2.5mM), sodium caprate (2-1graM) and interferon-gamma (IFN-/)(O.1- lOng/ml). EGTA treatment resulted in a complete loss of size-discrimination due to an increase in the radius of the restrictive pore. In contrast, sodium caprate and IFN~/had little effect on restrictive pore radius but appeared to increase the number of functional pores. IFN-/markedly increased the abundance of the larger,8 pores which correlated with an increased permeability to high MW dextrans. Conclusions: PEG profiling is a useful tool for functional modeling of the TJ. Preliminary studies using this technique suggest that some TJ modulators may not act by simple dilation of the TJ. This work was funded by AstraZeneca.

3781

Intestinal Barrier Function Can Be Regulated by DisUuct Mechauisms Modulating Transmembrane or Intracellular Components of Tight Junctions (T J). Takanori Sakaguchi, Raisuke Nishiyama, Tetsushi Kinugasa, Xiubin Go, MA Gen Hosp, Boston, MA; Richard P. Macdermott, Albany Medical Coil, Albany, NY; Daniel K. Podolsky, Hans-Christian Reinecker, MA Gen Hosp, Boston, MA

Background: Claudins and occludin have been identified as regulatory tight junction specific integral-membrane proteins. Both interact with the PDZ domains of ZO-1 and ZO-2, which cross-link TJ strands and actin filaments. To determine the regulation of TJ formation mediated by cytokine receptors in intestinal epithelial cells (lEO), we characterized basal and IL-15 mediated regulation of TJ associated proteins during the development of intestinal barrier function in model epithelia. Methods: T-84 cells or T-84,8 cells, which were stable transfected to over express IL-2R,8, were cultured on permeable filter support or cell culture dishes without or in the presence of IL-15 for four days after reaching confluence. Transepithelial electrical resistance (TER) and mannitol tracer flux characterized IEC barrier function. Expres- sion and membrane association of claudin-1, claudin-2, phosphorylated and non-phosphory- lated occludin, ZO-1, and ZO-2 were determined in Triton-X-lO0 soluble and insoluble protein fractions by Western blotting and by confocal microscopy. Results: The enhanced recruitment of tight junction proteins into Triton-X-lO0 insoluble protein fractions correlated with the tightening of the IEC monolayers in response to IL-15. Claudin-1 and claudin-2 localized into different cellular compartments in undifferentiated T-84 and T-84,8 cells. Whereas claudin-1 was mostly detected with ZO-2 in soluble protein fractions, claudin-2 was highly enriched in tight junctional protein fractions characterized by the expression of hyper-phosphorylated occludin and ZO-1. In the presence of IL-2R,8, IL-15 stimulated the phosphorylation of occludin and the recruitment of clauitin-1 and claudin-2 into insoluble protein fractions. In contrast, IL-15 mediated up-regulation of ZO-1 and ZO-2 expression was independent of the IL-2R,8 subunit. Recruitment of claudins and hyper-phosphorylated occludin into TJ resulted in a more marked increase of TER in IEC, than the up-regulation of ZO-1 and ZO-2 alone. Conclu- sions: Claudin-1 and claudin-2 may have distinct functions during the formation of tight junctional complexes in lEG. Cytokines are able to regulate TJ formation by distinct signals, which can induce either the recruitment of MAGUK family members into tight junctions, or the enhanced expression, activation and membrane association of transmembrane tight junctional proteins.

3782

Enhanced Intestinal Epithelial Barrier Function with Polyunsaturated Fatty Acids and Myotibroblasts. Natalia Y. Cockcroft, Diane J. Stevenson, Chris J. Hawkey, Univ Hosp, Nottingham United Kingdom

INTRODUCTION: Epidemiological studies suggest that diets rich in unsaturated fatty acids are associated with a lower incidence of inflammatory bowel disease and colonic malignancy. Recent work suggests that sub epithelial myofibroblasts orchestrate many aspects of barrier function. We used a well differentiated colonic epithelial cell line (HCA7, clone 29) to investigate the effects of human colonic myofibrobtasts, the ~3 fatty acid ecosapentaenoic acid (EPA), the Q6 fatty acid, -~linoleic acid and their interaction on barrier function. METHODS: HCA7 cells ( approx 30O,OOO/ml) were seeded on the upper surface of Costar filters. In some experiments human colonic myofibroblast monolayers had been established on the inferior surface prior to HCA7 cells seeding. Cells were cultured in the presence or absence of EPA (concentration) or GLA (concentration). MTS test was used to monitor cell viability. RESULTS: Co-cultured myofibroblasts consistently accelerated establishment of a functional electrical barrier (505 _+ 45 versus 229 _+ 32 control, n = 6, at 3 days p=<O.O01 ) EPA and GLA had no effect on cell viability at concentrations up to lOSM. Like myofibroblasts, both GLA and EPA accelerated electrical barrier formation (716.3 _+ 65 ohms with GLA, 755 _+ 8.2 ohms with EPA versus 153 _+ 2.5 control, at 2 days, n = 6, p=<O.O01.). However, no synergy between fatty acids and myofibroblasts was evident. CONCLUSIONS: Unsaturated

fatty acids accelerate establishment of a functional electrical barrier in colonic epithelial cells, by a direct effect. Myofibroblasts have a similar influence, acting in a paracrine way. However there was no evidence of synergy between fatty acids and myofibroblasts.

3783

Protein Kinase C (PKC)-.81 Mediates EGF-Induced Protection of the Microtubule (MT) Cytoskeleton & Intestinal Barrier Function (BF) against Oxidant Injury All Banan, Jeremy Z. Fields, Yang Zhang, Ece Mutlu, All Keshavarzian, Rush Univ, Chicago, IL

Abnormal mucosal BF appears to be a key factor in the pathogenesis of a variety of GI inflammatory disorders including inflammatory bowel disease, IBD. We recently showed using monolayers of human intestinal (Caco-2) cells that 1) MT integrity is key to EGF-induced protection of BF against oxidant injury; 2) Protection requires general PKC activity; 3) PKC- ,81 is an abundant isoform in our wild type (WT) cells & is thus likely to be involved in EGF protection. Hypothesis: PKC-,81 is critical in EGF protection against oxidant injury. Oaco-2 cells were transfected to either stably over-express PKC-,81 or to inhibit its expression, & then pre-incubated with low or high doses of EGF (1 or 10 ng/ml) or OAG, a PKC activator (0.01 or 50 uM) before oxidant (0.5 mM H202). Outcome measures were: BF (fluorometry), MT cytoskeletal stability (high resolution laser scanning confocal; immunoblotting), PKC-,81 subcellular distribution (immunoblotting) as well as tubulin (structural protein of MT) assembly & disassembly (quantitative western), n =6/group. In WT cells, pretreatment with high doses of EGE or OAG protected against H202-induced disruption of MT & BF while translocating PKC-,81 to membrane fractions (i.e., activation); low doses did not protect. In transfected cells stably overexpressing PKC-,81 (-3.1 fold ), pretreatment with even low doses of EGF or OAG synergized to: polymerized tubulin & ) monomeric tubulin; integrity of MT cytoskeleton; maintain monolayer BF. This protection by low doses of EGF or OAG did not occur in wr cells. Over-expressed PKC-/~I was found in the cytosolic (C), membrane (M), & cytoskeletal (1) fractions of transfected cells. Pretreatment with EGF or OAG caused a rapid shift in distribution of over-expressed PKC-,81 into the M & T fractions concomitant with a ) in its C pool, indicating PKC-,81 activation. Transfestion of anti-sense to stably inhibit PKC- ,81 protein expression (90% }) abolished EGF-induced protection as indicated by: ) in polymerized tubulin & in depolymerized tubulin; instability of MT; monolayer hyperpermeabil- ity. We show for the first time that 1) EGF-induced protection of the MT cytoskeleton & of intestinal barrier integrity is mediated, at least in large part, through PKC-,81 isoform. 2) PKC- ,81 isoform is a key intracellular regulator of epithelial BE. 3) PKC-,81 signaling pathways may be useful as targets for development of novel therapies for the treatment of a variety of oxidative inflammatory disorders such as IBD.

3784

IFN~-induced Translocation Across T84 Mooolayers Is Dissociated From Paracellular Permeability Edwin C. Clark, Chris J. Watson, Univ of Manchester, Manchester United Kingdom; Gordon L. Carlson, Hope Hosp, Salford United Kingdom; Geoff Warhurst, Univ of Manchester, Manchester United Kingdom

Background: Translocation of commensal gut bacteria across the intact gut wall in critical illness is associated with sepsis. The route by which bacteria cross the gut epithelium remains poorly defined. Pro-inflammatory cytokines, which are known to modulate tight junctions, are increased during the early stages of critical illness. Aims: The aims of this study were therefore to investigate the effects of interferon gamma (IFN~,) on bacterial translocation across T84 cell monolayers and to examine the correlation between translocation and changes in paracellular permeability. Methods: T84 cells grown for 21 days on Transwell TM membranes were incubated on the basolateral surface with IFN'y (O.01-10ng/ml)for 48hr. Paracellular permeability of IFN.y-treated monolayers was measured using the fluorescent dye lucifer yellow (LY)and polyethylene glycol (PEG)oligomers of incresaing hydrodynamic radius (0.35- O.74nm) measured by LC-MS (Watson st a12000). Bacterial translocation was measured after inoculation of apical chambers with E.coli C25 (108 CFU)and serial dilution culture of samples from the basolateral chamber over a 4hr period. Results are expressed as mean + SD for 6 determinations in each group. Results: IFN,y stimulated a dose dependent increase increase in bacterial translocation (Table) with a threshold for translocation of O.lng/ml. IFN'y also caused a marked increase in both LY and PEG permeability but the threshold for these effects occurred at a lO-fold higher concentration (lng/ml). Conclusions: IFN,,/-induced bacterial translocation across T84 monolayers is differentiated from increases in paracellular permeabil- ity. Bacterial translocation across the gut in inflammatory conditions may occur via a transcellu- lar route.

Effect of IFNy on bacterial t r ans l~n , LY & PEG permeability across T84

[IFNy] ng/ml [basal bacteria] [lucifer yellow] as 0.376nm PEG CFUlml % apical Papp(cm/s)

O 3.9±0.3 1.3±0.4 0.19±0.04 0.1 5.9±0.2 ~ 1.4±0.4 0.19-J:0.02 t 5.7±02 ** 8.7+_0.5 ** 6.40!-_0.04"* to 6.5_+0.2 ** t5.7±1.3" 0.46~0.05 **

= P <0.001 unpaired Student-t test (control Ong/ml IFN';,)

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