Jan F. Richter

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.

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.

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.

Mechanisms of epithelial translocation of the α2-gliadin-33mer in coeliac sprue

Background and aims: The α2-gliadin-33mer has been shown to be important in the pathogenesis of coeliac disease. We aimed to study mechanisms of its epithelial translocation and processing in respect to transcytotic and paracellular pathways. Methods: Transepithelial passage of a fluorescence-labelled α2-gliadin-33mer was studied in Caco-2 cells by using reverse-phase high-performance liquid chromatography, mass spectrometry, confocal laser scanning microscopy (LSM) and fluorescence activated cell sorting (FACS). Endocytosis mechanisms were characterised with rab-GFP constructs transiently transfected into Caco-2 cells and in human duodenal biopsy specimens. Results: The α2-gliadin-33mer dose-dependently crossed the epithelial barrier in the apical-to-basal direction. Degradation analysis revealed translocation of the 33mer polypeptide in the uncleaved as well as in the degraded form. Transcellular passage was identified by confocal LSM, inhibitor experiments and FACS. Rab5 but not rab4 or rab7 vesicles were shown to be part of the transcytotic pathway. After pre-incubation with interferon-γ, translocation of the 33mer was increased by 40%. In mucosal biopsies of the duodenum, epithelial 33mer uptake was significantly higher in untreated coeliac disease patients than in healthy controls or coeliac disease patients on a gluten-free diet. Conclusion: Epithelial translocation of the α2-gliadin-33mer occurs by transcytosis after partial degradation through a rab5 endocytosis compartment and is regulated by interferon-γ. Uptake of the 33mer is higher in untreated coeliac disease than in controls and coeliac disease patients on a gluten-free diet.

Fermentable Fiber Ameliorates Fermentable Protein-Induced Changes in Microbial Ecology, but Not the Mucosal Response, in the Colon of Piglets

Dietary inclusion of fermentable carbohydrates (fCHO) is reported to reduce large intestinal formation of putatively toxic metabolites derived from fermentable proteins (fCP). However, the influence of diets high in fCP concentration on epithelial response and interaction with fCHO is still unclear. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO [14.5% crude protein (CP)/14.5% total dietary fiber (TDF)]; low fCP/high fCHO (14.8% CP/16.6% TDF); high fCP low fCHO (19.8% CP/14.5% TDF); and high fCP/high fCHO (20.1% CP/18.0% TDF) as dietary treatments. After 21-23 d, pigs were killed and colon digesta and tissue samples analyzed for indices of microbial ecology, tissue expression of genes for cell turnover, cytokines, mucus genes (MUC), and oxidative stress indices. Pig performance was unaffected by diet. fCP increased (P < 0.05) cell counts of clostridia in the Clostridium leptum group and total short and branched chain fatty acids, ammonia, putrescine, histamine, and spermidine concentrations, whereas high fCHO increased (P < 0.05) cell counts of clostridia in the C. leptum and C. coccoides groups, shifted the acetate to propionate ratio toward acetate (P < 0.05), and reduced ammonia and putrescine (P < 0.05). High dietary fCP increased (P < 0.05) expression of PCNA, IL1β, IL10, TGFβ, MUC1, MUC2, and MUC20, irrespective of fCHO concentration. The ratio of glutathione:glutathione disulfide was reduced (P < 0.05) by fCP and the expression of glutathione transferase was reduced by fCHO (P < 0.05). In conclusion, fermentable fiber ameliorates fermentable protein-induced changes in most measures of luminal microbial ecology but not the mucosal response in the large intestine of pigs.

Cell polarity-determining proteins Par-3 and PP-1 are involved in epithelial tight junction defects in coeliac disease

Background Epithelial barrier defects are well known in coeliac disease, but the mechanisms are only poorly defined. It is unclear, whether barrier disturbance reflects upregulated epithelial transcytosis or paracellular leakage. Objective To characterise the molecular structure and function of the epithelial tight junction (TJ) and mechanisms of its dysregulation. Methods Molecular analysis of proteins involved in TJ assembly and their regulation was performed by western blotting and confocal microscopy correlated to electrophysiology. Results A complex alteration of the composition of epithelial TJ proteins (with more pore-forming claudins like claudin-2 and a reduction in tightening claudins like claudin-3, -5 and -7) was found for protein expression and subcellular localisation, responsible for an increase in paracellular biotin-NHS uptake. In contrast, epithelial apoptosis was only moderately elevated (accounting for a minor portion of barrier defects) and epithelial gross lesions—for example, at cell extrusion zones, were absent. This TJ alteration was linked to an altered localisation/expression of proteins regulating TJ assembly, the polarity complex protein Par-3 and the serine-/threonine phosphatase PP-1. Conclusions Changes in cell polarity proteins Par-3 and PP-1 are associated with altered expression and assembly of TJ proteins claudin-2, -3, -5 and -7 and ZO-1, causing paracellular leakage in active coeliac disease.

Escherichia coli alpha-haemolysin induces focal leaks in colonic epithelium: a novel mechanism of bacterial translocation

Extraintestinal pathogenic Escherichia coli (ExPEC) are usually harmless colonizer of the intestinal microflora. However, they are capable to translocate and cause life‐threatening disease. Translocation of ExPEC isolates was quantified in colonic monolayers. Transepithelial resistance (Rt) was monitored and local changes in conductivity analysed with conductance scanning. Confocal microscopy visualized the translocation route. Corroboratory experiments were performed on native rat colon. One translocating strain E. coli O4 was identified. This translocation process was associated with an Rt decrease (36 ± 1% of initial resistance) beginning only 2 h after inoculation. The sites of translocation were small defects in epithelial integrity (focal leaks) exhibiting highly increased local ion permeability. Translocation was enhanced by preincubation of monolayers with tumour necrosis factor‐α or interleukin‐13. Mutant strains lacking alpha‐haemolysin lost the ability to induce focal leaks, while this effect could be restored by re‐introducing the haemolysin determinant. Filtrate of a laboratory strain carrying the alpha‐haemolysin operon was sufficient for focal leak induction. In native rat colon, E. coli O4 decreased Rt and immunohistology demonstrated focal leaks resembling those in cell monolayers. E. coliα‐haemolysin is able to induce focal leaks in colonic cell cultures as well as in native colon. This process represents a novel route of bacterial translocation facilitated by pro‐inflammatory cytokines.

Claudin-16 affects transcellular Cl − secretion in MDCK cells

Claudin‐16 (paracellin‐1) is a tight junction protein localized mainly in the thick ascending limb of Henles loop and also in the distal nephron. Its defect causes familial hypomagnesaemia with hypercalciuria and nephrocalcinosis. This had been taken as an indication that claudin‐16 conveys paracellular Mg2+ and Ca2+ transport; however, evidence is still conflicting. We studied paracellular ion permeabilties as well as effects of claudin‐16 on the driving forces for passive ion movement. MDCK‐C7 cells were stably transfected with wild‐type (wt) and mutant (R146T, T233R) claudin‐16. Results indicated that paracellular permeability to Mg2+ but not to Ca2+ is increased in cells transfected with wt compared to mutant claudin‐16 and control cells. Increased basolateral Mg2+ concentration activated a transcellular Cl− current which was greatly enhanced in cells transfected with wt and T233R claudin‐16, as compared to R146T claudin‐16‐transfected or control cells. This current was triggered by the basolateral calcium‐sensing receptor causing Ca2+ release from internal stores, thus activating apical Ca2+‐sensitive Cl− channels and basolateral Ca2+‐sensitive K+ channels. Immunohistochemical data suggest that the Cl− channel involved is bestrophin. We conclude that claudin‐16 itself possesses only moderate paracellular Mg2+ permeability but governs transcellular Cl− currents by interaction with apical Ca2+‐activated Cl− channels, presumably bestrophin. As the transepithelial voltage generated by such a current alters the driving force for all ions, this may be the major mechanism to regulate Mg2+ and Ca2+ absorption in the kidney.

Inhibition of Inflammatory Pain by CRF at Peripheral, Spinal and Supraspinal Sites: Involvement of Areas Coexpressing CRF Receptors and Opioid Peptides

There is conflicting evidence on the antinociceptive effects of corticotropin-releasing factor (CRF) along the neuraxis of pain transmission and the responsible anatomical sites of CRFs action at the level of the brain, spinal cord and periphery. In an animal model of tonic pain, that is, Freunds complete adjuvant (FCA) hindpaw inflammation, we systematically investigated CRFs ability to modulate inflammatory pain at those three levels of pain transmission by algesiometry following the intracerebroventricular, intrathecal, and intraplantar application of low, systemically inactive doses of CRF. At each level, CRF elicits potent antinociceptive effects, which are dose dependent and antagonized by local, but not systemic CRF receptor antagonist α-helical CRF indicating CRF receptor specificity. Consistently, we have identified by immunohistochemistry multiple brain areas, inhibitory interneurons within the dorsal horn of the spinal cord as well as immune cells within subcutaneous tissue—but not peripheral sensory neurons—that coexpress both CRF receptors and opioid peptides. In line with these anatomical findings, local administration of CRF together with the opioid receptor antagonist naloxone dose-dependently reversed CRFs antinociceptive effects at each of these three levels of pain transmission. Therefore, local application of low, systemically inactive doses of CRF at the level of the brain, spinal cord and periphery inhibits tonic inflammatory pain most likely through an activation of CRF receptors on cells that coexpress opioid peptides which results in opioid-mediated pain inhibition. Future studies have to delineate whether endogenous CRF at these three levels contributes to the bodys response to cope with the stressful stimulus pain in an opioid-mediated manner.

Improved Cell Line IPEC-J2, Characterized as a Model for Porcine Jejunal Epithelium

Cell lines matching the source epithelium are indispensable for investigating porcine intestinal transport and barrier properties on a subcellular or molecular level and furthermore help to reduce animal usage. The porcine jejunal cell line IPEC-J2 is established as an in vitro model for porcine infection studies but exhibits atypically high transepithelial resistances (TER) and only low active transport rates so that the effect of nutritional factors cannot be reliably investigated. This study aimed to properly remodel IPEC-J2 and then to re-characterize these cells regarding epithelial architecture, expression of barrier-relevant tight junction (TJ) proteins, adequate TER and transport function, and reaction to secretagogues. For this, IPEC-J2 monolayers were cultured on permeable supports, either under conventional (fetal bovine serum, FBS) or species-specific (porcine serum, PS) conditions. Porcine jejunal mucosa was analyzed for comparison. Main results were that under PS conditions (IPEC-J2/PS), compared to conventional FBS culture (IPEC-J2/FBS), the cell height increased 6-fold while the cell diameter was reduced by 50%. The apical cell membrane of IPEC-J2/PS exhibited typical microvilli. Most importantly, PS caused a one order of magnitude reduction of TER and of trans- and paracellular resistance, and a 2-fold increase in secretory response to forskolin when compared to FBS condition. TJ ultrastructure and appearance of TJ proteins changed dramatically in IPEC-J2/PS. Most parameters measured under PS conditions were much closer to those of typical pig jejunocytes than ever reported since the cell line’s initial establishment in 1989. In conclusion, IPEC-J2, if cultured under defined species-specific conditions, forms a suitable model for investigating porcine paracellular intestinal barrier function.