Michael Fromm

Changes in expression and distribution of claudin-2, -5 and -8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease

Background: Epithelial barrier function is impaired in Crohn’s disease. Aim: To define the underlying cellular mechanisms with special attention to tight junctions. Methods: Biopsy specimens from the sigmoid colon of patients with mild to moderately active or inactive Crohn’s disease were studied in Ussing chambers, and barrier function was determined by impedance analysis and conductance scanning. Tight junction structure was analysed by freeze fracture electron microscopy, and tight junction proteins were investigated immunohistochemically by confocal laser scanning microscopy and quantified in immunoblots. Epithelial apoptosis was analysed in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and 4′,6-diamidino-2-phenylindole staining. Results: Patients with active Crohn’s disease showed an impaired intestinal barrier function as indicated by a distinct reduction in epithelial resistance. As distribution of conductivity was even, focal epithelial lesions (eg, microerosions) did not contribute to barrier dysfunction. Instead, freeze fracture electron microscopy analysis showed reduced and discontinuous tight junction strands. Occludin and the sealing tight junction proteins claudin 5 and claudin 8 were downregulated and redistributed off the tight junction, whereas the pore-forming tight junctions protein claudin 2 was strongly upregulated, which constitute the molecular basis of tight junction changes. Other claudins were unchanged (claudins 1, 4 and 7) or not detectable in sigmoid colon (claudins 11, 12, 14, 15 and 16). Claudin 2 upregulation was less pronounced in active Crohn’s disease compared with active ulcerative colitis and was inducible by tumour necrosis factor α. As a second source of impaired barrier function, epithelial apoptosis was distinctly increased in active Crohn’s disease (mean (SD) 5.2 (0.5)% v 1.9 (0.2)% in control). By contrast, barrier function, tight junction proteins and apoptosis were unaffected in Crohn’s disease in remission. Conclusion: Upregulation of pore-forming claudin 2 and downregulation and redistribution of sealing claudins 5 and 8 lead to altered tight junction structure and pronounced barrier dysfunction already in mild to moderately active Crohn’s disease.

Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis

BACKGROUND & AIMS Mechanisms of diarrhea in ulcerative colitis (UC) are still unknown. Functional and structural characterization of epithelial barrier and transport properties in ulcerative colitis (UC) was performed. METHODS Inflamed sigmoid colon epithelium from UC patients was studied by alternating current impedance analysis to determine the pure epithelial resistance as a measure of intestinal barrier function. Tight junction (TJ) structure was investigated by freeze-fracture electron microscopy. RESULTS Although total wall resistance was reduced in UC by 50%, impedance analysis uncovered a much more pronounced barrier defect. Epithelial resistance decreased from 95 +/- 5 to 20 +/- 3 omega3. cm2, which in conventional analysis is masked by an increase in subepithelial resistance from 14 +/- 1 to 36 +/- 3 omega3. cm2 caused by inflammation. This was paralleled by a change in epithelial cell TJ structure in UC. Strand count decreased from 6.94 +/- 0.25 to 4.76 +/- 0.47 at the surface and from 7.26 +/- 0.31 to 5.46 +/- 0.37 in the crypts. CONCLUSIONS The inflamed colonic mucosa in UC has an impaired barrier function that is much more pronounced than previously assumed. An altered TJ structure contributes to this barrier defect which, because of increased back leak, can reduce net ion transport. Thus, a leak-flux mechanism contributes to the diarrhea in UC.

E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions

Cadherin adhesion molecules are key determinants of morphogenesis and tissue architecture. Nevertheless, the molecular mechanisms responsible for the morphogenetic contributions of cadherins remain poorly understood in vivo. Besides supporting cell–cell adhesion, cadherins can affect a wide range of cellular functions that include activation of cell signalling pathways, regulation of the cytoskeleton and control of cell polarity. To determine the role of E‐cadherin in stratified epithelium of the epidermis, we have conditionally inactivated its gene in mice. Here we show that loss of E‐cadherin in the epidermis in vivo results in perinatal death of mice due to the inability to retain a functional epidermal water barrier. Absence of E‐cadherin leads to improper localization of key tight junctional proteins, resulting in permeable tight junctions and thus altered epidermal resistance. In addition, both Rac and activated atypical PKC, crucial for tight junction formation, are mislocalized. Surprisingly, our results indicate that E‐cadherin is specifically required for tight junction, but not desmosome, formation and this appears to involve signalling rather than cell contact formation.

Downregulation of epithelial apoptosis and barrier repair in active Crohn’s disease by tumour necrosis factor α antibody treatment

Background and aims: Barrier dysfunction is an important feature contributing to inflammation and diarrhoea in Crohn’s disease (CD). Recently, tumour necrosis factor α (TNF-α) antibodies were recognised as effective in steroid refractory CD. The aim of this study was to characterise the effects of this therapy on the epithelial barrier. Patients and methods: Forceps biopsies were obtained from the sigmoid colon before and 14 days after TNF-α antibody therapy in 11 patients treated for chronic active CD (Crohn’s disease activity index >150). Epithelial apoptoses were measured after terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) and 4′,6-diamidino-2-phenylindole staining. Epithelial resistance was determined by alternating current impedance analysis in miniaturised Ussing chambers. Occludin, claudin 1, and claudin 4 expression was quantified in immunoblots. Results: The epithelial apoptotic ratio was 2.1 (0.2)% in controls and increased to 5.3 (1.0)% in CD. TNF-α antibody therapy decreased the apoptotic ratio to 2.9 (1.0)% (normalised in 10 of 11 patients). In parallel, epithelial resistance was lower in CD than in controls (24 (3) v 42 (3) Ω×cm2) and improved to 34 (3) Ω×cm2 after therapy. Occludin, claudin 1, and claudin 4 were not affected by TNF-α antibody therapy. In support of a functional role of epithelial apoptoses in CD, a similar decrease in resistance of −40% was observed when the apoptotic rate was selectively upregulated from 2.6% to 5.4% with camptothecin in HT-29/B6 cells. Conclusions: Epithelial apoptoses were upregulated in the colon in CD and restored to normal in 10 of 11 patients by TNF-α antibody therapy. This is the structural correlate of epithelial barrier dysfunction measured as epithelial resistance while expression of tight junction proteins did not contribute to this therapeutic effect.

Epithelial Tight Junctions in Intestinal Inflammation

The epithelium in inflamed intestinal segments of patients with Crohns disease is characterized by a reduction of tight junction strands, strand breaks, and alterations of tight junction protein content and composition. In ulcerative colitis, epithelial leaks appear early due to micro‐erosions resulting from upregulated epithelial apoptosis and in addition to a prominent increase of claudin‐2. Th1‐cytokine effects by interferon‐γ in combination with TNFα are important for epithelial damage in Crohns disease, while interleukin‐13 (IL‐13) is the key effector cytokine in ulcerative colitis stimulating apoptosis and upregulation of claudin‐2 expression. Focal lesions caused by apoptotic epithelial cells contribute to barrier disturbance in IBD by their own conductivity and by confluence toward apoptotic foci or erosions. Another type of intestinal barrier defect can arise from α‐hemolysin harboring E. coli strains among the physiological flora, which can gain pathologic relevance in combination with proinflammatory cytokines under inflammatory conditions. On the other hand, intestinal barrier impairment can also result from transcellular antigen translocation via an initial endocytotic uptake into early endosomes, and this is intensified by proinflammatory cytokines as interferon‐γ and may thus play a relevant role in the onset of IBD. Taken together, barrier defects contribute to diarrhea by a leak flux mechanism (e.g., in IBD) and can cause mucosal inflammation by luminal antigen uptake. Immune regulation of epithelial functions by cytokines may cause barrier dysfunction not only by tight junction impairments but also by apoptotic leaks, transcytotic mechanisms, and mucosal gross lesions.

Claudin-2, a component of the tight junction, forms a paracellular water channel

Whether or not significant amounts of water pass the tight junction (TJ) of leaky epithelia is still unresolved, because it is difficult to separate transcellular water flux from TJ-controlled paracellular water flux. Using an approach without differentiating technically between the transcellular and paracellular route, we measured transepithelial water flux with and without selective molecular perturbation of the TJ to unequivocally attribute changes to the paracellular pathway. To this end, MDCK C7 cells were stably transfected with either claudin-2 or claudin-10b, two paracellular cation-channel-forming TJ proteins that are not endogenously expressed in this cell line. Claudin-2 is typical of leaky, water-transporting epithelia, such as the kidney proximal tubule, whereas claudin-10b is present in numerous epithelia, including water-impermeable segments of the loop of Henle. Neither transfection altered the expression of endogenous claudins or aquaporins. Water flux was induced by an osmotic gradient, a Na+ gradient or both. Under all conditions, water flux in claudin-2-transfected cells was elevated compared with vector controls, indicating claudin-2-mediated paracellular water permeability. Na+-driven water transport in the absence of an osmotic gradient indicates a single-file mechanism. By contrast, claudin-10b transfection did not alter water flux. We conclude that claudin-2, but not claudin-10b, forms a paracellular water channel and thus mediates paracellular water transport in leaky epithelia.

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

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 α (TNF‐α) 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‐α‐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‐α‐induced single‐cell apoptosis. FASEB J. 14, 1749–1753 (2000)

Mechanisms of diarrhea in collagenous colitis

BACKGROUND & AIMS Collagenous colitis is an inflammatory disease of unknown etiology with diarrhea as the leading symptom. The aim of this study was to examine the pathogenic mechanisms of this disease. METHODS Biopsy specimens of the sigmoid colon were obtained endoscopically. Short-circuit current and (22)Na and (36)Cl fluxes were measured in miniaturized Ussing chambers. Alternating current impedance analysis discriminated epithelial from subepithelial resistance. Tight junction proteins occludin and claudin 1-5 were characterized in membrane fractions by Western blotting. Apoptotic ratio was determined by DAPI and TUNEL staining. RESULTS In collagenous colitis, net Na(+) flux decreased from 8.8 +/- 1.8 to 0.2 +/- 1.5 and net Cl(-) flux from 11.2 +/- 3.0 to -3.0 +/- 2.7 micromol x h(-1) x cm(-2), indicating a pronounced decrease in NaCl absorption. The fact that short-circuit current increased from 1.5 +/- 0.4 to 3.9 +/- 0.8 micromol x h(-1) x cm(-2), together with the negative net Cl(-) flux, points to activation of active electrogenic chloride secretion. Subepithelial resistance increased from 7 +/- 1 to 18 +/- 2 Omega x cm(2) due to subepithelial collagenous bands of 48 +/- 8-microm thickness. Epithelial resistance was diminished from 44 +/- 3 to 29 +/- 2 Omega x cm(2), and this was accompanied by a decrease in occludin and claudin-4 expression. Neither mucosal surface area nor apoptotic ratio was altered in collagenous colitis. CONCLUSIONS Reduced net Na(+) and Cl(-) absorption is the predominant diarrheal mechanism in collagenous colitis, accompanied by a secretory component of active electrogenic chloride secretion. The subepithelial collagenous band as a significant diffusion barrier is a cofactor. Down-regulation of tight junction molecules but not epithelial apoptoses is a structural correlate of barrier dysfunction contributing to diarrhea by a leak flux mechanism.

Molecular Basis for Cation Selectivity in Claudin-2―based Paracellular Pores: Identification of an Electrostatic Interaction Site

Paracellular ion transport in epithelia is mediated by pores formed by members of the claudin family. The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood. By expressing a high-conductance claudin isoform, claudin-2, in high-resistance Madin-Darby canine kidney cells under the control of an inducible promoter, we were able to quantitate claudin pore permeability. Claudin-2 pores were found to be narrow, fluid filled, and cation selective. Charge selectivity was mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.

Tricellulin Forms a Barrier to Macromolecules in Tricellular Tight Junctions without Affecting Ion Permeability

Tricellulin is a tight junction protein localized in tricellular tight junctions (tTJs), the meeting points of three cells, but also in bicellular tight junctions (bTJs). To investigate its specific barrier functions in bTJs and tTJs, TRIC-a was expressed in low-level tricellulin-expressing cells, and MDCK II, either in all TJs or only in tTJs. When expressed in all TJs, tricellulin increased paracellular electrical resistance and decreased permeability to ions and larger solutes, which are associated with enhanced ultrastructural integrity of bTJs toward enhanced strand linearity. In tTJs in contrast, ultrastructure was unchanged and tricellulin minimized permeability to macromolecules but not to ions. This paradox is explained by properties of the tTJ central tube which is wide enough for passage of macromolecules, but too rare to contribute significantly to ion permeability. In conclusion, at low tricellulin expression the tTJ central tube forms a pathway for macromolecules. At higher expression, tricellulin forms a barrier in tTJs effective only for macromolecules and in bTJs for solutes of all sizes.