Regulation of mucosal structure and barrier function in rat colon exposed to tumor necrosis factor alpha and interferon gamma in vitro: a novel model for studying the pathomechanisms of inflammatory bowel disease cytokines.
OBJECTIVE
In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal transport and barrier function. The mechanisms of cytokine action have usually been studied in cell culture models only; therefore the aim of this study was to establish an in vitro model based on native intestine to analyze distinct cytokine effects on barrier function, mucosal structure, and inherent regulatory mechanisms.


METHODS
Rat colon was exposed to tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) in Ussing chambers. Transepithelial resistance (R(t)) and (3)H-mannitol fluxes were measured for characterization of the paracellular pathway. Transcellular transport was analyzed by horseradish peroxidase (HRP) flux measurements. Expression and distribution of tight junction proteins were characterized in immunoblots and by means of confocal laser-scanning microscopy (LSM).


RESULTS
Colonic viability could be preserved for 20 h in a specialized in vitro set-up. This was sufficient to alter mucosal architecture with crypt surface reduction. R(t) was decreased (101+/-10 versus 189+/-10 Omega x cm(2)) with a parallel increase in mannitol permeability after cytokine exposure. Tight junction proteins claudin-1, -5, -7, and occludin decreased (45+/-10%, 16+/-7%, 42+/-8%, and 42+/-13% of controls, respectively), while claudin-2 increased to 208+/-32%. Occludin and claudin-1 translocated from the plasma membrane to the cytoplasm. HRP flux increased from 0.73+/-0.09 to 8.55+/-2.92 pmol x h(-1) x cm(-2).


CONCLUSIONS
A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFalpha and IFNgamma alters mucosal morphology and impairs epithelial barrier function by up-regulation of the paracellular pore-former claudin-2 and down-regulation of the barrier-builders claudin-1, -5, and -7. These alterations resemble changes seen in IBD and thus underline their prominent role in IBD pathogenicity.
Amasheh
Maren
M
Department of Medicine I-Gastroenterology, Infectious Diseases and Rheumatology, Charité, Campus Benjamin Franklin, Berlin, Germany.
Grotjohann
Ingo
I
Amasheh
Salah
S
Fromm
Anja
A
Söderholm
Johan D
JD
Zeitz
Martin
M
Fromm
Michael
M
Schulzke
Jörg-Dieter
JD
D000818:Animals; D004195:Disease Models, Animal; D066298:In Vitro Techniques; D015212:Inflammatory Bowel Diseases / Q000276:immunology / Q000503:physiopathology*; D007371:Interferon-gamma / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000503:physiopathology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma