Biomedical subjects

Christian Bojarski

Publications and source records attributed to Christian Bojarski.

4 recordsLinked to original sources

PubMed2006

Disrupted barrier function through epithelial cell apoptosis.

Epithelial barrier function is determined by trans- and paracellular permeabilities, the latter of which is mainly influenced by tight junctions (TJs) and apoptotic leaks within the epithelium. The present article aims to present experimental evidence for a functional role of epithelial apoptoses by means of cell culture models as well as in tissues from patients with inflammatory bowel disease. It is shown that epithelial apoptoses are sites of elevated conductance within the intestinal epithelium and that proinflammatory cytokines like TNF-alpha upregulate both the apoptotic rate and single apoptotic conductivity, making cytokine-induced apoptosis functionally far more relevant than is spontaneous apoptosis. In ulcerative colitis and Crohn's disease (CD), but not in collagenous colitis, apoptotic rates are increased to about 5%, in mild-to-moderately inflamed colon specimens, where as the control apoptotic rate is about 2%. Thus, epithelial apoptoses lead to a loss of ions and water into the intestinal lumen, causing leak flux diarrhea and enabling small antigens of <4,000 Da in the intestinal lumen to enter the intestinal mucosa, thereby perpetuating inflammatory responses. In addition to TNF-alpha, interleukin (IL)-13 is an important inductor of epithelial apoptosis in Th2 immune responses. Therapeutically,TNF-alpha-antibodies (infliximab) can restore barrier function in Crohn's disease by downregulating epithelial apoptoses, while epithelial TJs are unaffected.

Apoptosis↗

PubMed2005

Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions, apoptosis, and cell restitution.

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by a Th2 immune response with inflammation and epithelial barrier dysfunction. So far, Th2 cytokines have not been shown to directly influence epithelial barrier function. METHODS: Lamina propria mononuclear cells (LPMCs) were stimulated and interleukin (IL)-13 was measured by enzyme-linked immunosorbent assay. Functional IL-13 and IL-4 effects were studied on HT-29/B6 colonic epithelial cells in Ussing chambers and by conductance scanning. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. IL-13/IL-4 receptors were analyzed by reverse-transcription polymerase chain reaction and immunofluorescence. Western blotting combined with immunofluorescence was used to detect tight junction proteins. Furthermore, restitution velocity was measured. Finally, mucosal biopsy specimens from patients with UC were compared with cultured cells for these features. RESULTS: LPMCs from patients with UC produced large amounts of IL-13 (985 +/- 73 pg/mL), much more than from controls or patients with Crohn's disease. IL-13Ralpha1 and IL-4Ralpha receptors were present in HT-29/B6 cells and colonic epithelial cells of control patients and patients with UC. IL-13 had a dose-dependent effect on transepithelial resistance of HT-29/B6 monolayers (reduction to 60% +/- 4%), whereas IL-4 had no effect. This was due to an increased number of apoptotic cells (5.6-fold +/- 0.9-fold) and an increased expression of the pore-forming tight junction protein claudin-2 to 295% +/- 37%, both of which contributed equally. Finally, epithelial restitution velocity decreased from 15.1 +/- 0.6 to 10.6 +/- 0.5 microm/h after treatment with IL-13. Parallel changes were observed in human samples, with an increase in claudin-2 expression to 956% +/- 252%. CONCLUSIONS: IL-13 was identified as an important effector cytokine in UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity.

Adult↗

PubMed2004

The specific fates of tight junction proteins in apoptotic epithelial cells.

The polarized morphology of epithelial cells depends on the establishment and maintenance of characteristic intercellular junctions. The dramatic morphological changes observed in apoptotic epithelial cells were ascribed at least in part to the specific fragmentation of components of adherens junctions and desmosomes. Little, however, is known about tight junctions during apoptosis. We have found that after induction of apoptosis in epithelial cells, tight junction proteins undergo proteolytic cleavage in a distinctive manner correlated with a disruption of tight junctions. The transmembrane protein occludin and, likewise, the cytoplasmic adaptor proteins ZO-1 and ZO-2 are fragmented by caspase cleavage. In addition, occludin is cleaved at an extracellular site by a metalloproteinase. The caspase cleavage site in occludin was mapped C-terminally to Asp(320) within the C-terminal cytoplasmic domain. Mutagenesis of this site efficiently blocked fragmentation. In the presence of caspase and/or metalloproteinase inhibitors, fragmentation of occludin, ZO-1 and ZO-2 was blocked and cellular morphology was almost fully preserved. Interestingly, two members of the claudin family of transmembrane tight junction proteins exhibited a different behavior. While the amount of claudin-2 protein was reduced similarly to occludin, ZO-1 and ZO-2, claudin-1 was either fully preserved or was even increased in apoptotic cells.

Animals↗

PubMed2002

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.

Adult↗