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

Abstract

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 49,6-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 poreforming 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 a. 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.

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

“…In our TRIC-a overexpression experiments the opposite occurred in bTJs: Although the meshwork depth and the strand number remained constant in comparison to controls, the occurrence of breaks Ͼ20 nm was decreased, and strand type changed to 100% continuous. A similar correlation between strand linearity and paracellular resistance was found in human colon to be induced by a strong upregulation of claudin-2 in Crohn's disease (Zeissig et al, 2007). It is established that, depending on the presence of claudin-2, strand discontinuities appear or disappear (Furuse et al, 1999).…”

“…Freeze-fracture electron microscopy was performed as previously described (Zeissig et al, 2007). Briefly, cells grown on permeable supports were fixed with phosphate-buffered glutaraldehyde (2%).…”

Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function

“…However, when the epithelial barrier was functionally assessed, we demonstrated decreased TEER, increased permeability to a 4 kDa paracellular probe and confirmed that Claudin-2, a known pore forming TJ protein, was significantly increased at the protein level in decompensated cirrhosis, suggesting that it is responsible for the increased permeability observed. Indeed, epithelial barrier dysfunction and elevated Claudin-2 expression associated with bacterial translocation have been observed in other diseases, such as Crohn's disease [26] and HIV infection [27]. Despite demonstrating increased permeability, it remains unclear to what extent Claudin-2 induced pore formation allows crossing of large bacterial products such as LPS and DNA.…”

Neutrophil-Epithelial Interactions

“…23 Similar alterations in claudin family members have been described under conditions of inflammation, as seen in the intestinal mucosa of individuals with IBD. 98,99 Furthermore, selective disruption of specific subsets of tight junction proteins can be seen after exposure to inflammatory cytokines. As an example, Figure 5A highlights the effect of exposing cultured epithelial monolayers to the cytokine interferon-g that results in selective disorganization of several tight junction proteins while preserving architecture for the adherens junction proteins E-cadherin and b-catenin.…”