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North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Annual Meeting, October 19-22, 2006, Orlando, Florida: Abstracts: ORAL ABSTRACT PRESENTATIONS: HEPATOLOGY/IBD/CELIAC DISEASE SATURDAY, OCTOBER 21, 2006, 10 a.m.- 12 Noon: 187

GLIADIN BINDING TO CXCR3 CAUSES ZONULIN RELEASE AND INCREASED INTESTINAL PERMEABILITY

Lammers, Karen1; Brownley, Julie1; Lu, Ruiliang1; Sapone, Anna1; Lu, Bao2; Gerard, Craig2; Vogel, Stefanie1; Fasano, Alessio1

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Journal of Pediatric Gastroenterology and Nutrition: October 2006 - Volume 43 - Issue 4 - p E72
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Background:

The Celiac Disease (CD) autoimmune process is triggered by gliadin ingestion. Our previous studies showed that gliadin triggers the release of zonulin, a modulator of intestinal permeability (IP) only when gliadin was added to the mucosal side of the intestine.

Aim:

To establish whether the effect of gliadin on zonulin release and IP is mediated by the interaction of gliadin with an intestinal luminal receptor and to investigate the identity of this receptor.

Methods:

Soluble membrane preparations were obtained from rabbit small intestine and loaded on an Affi-gel 15-alfa-gliadin affinity column. Collected fractions were subjected to SDS-PAGE and mass spectroscopy analysis. In vitro experiments using CXCR3-transfected HEK293T cells were performed to study gliadin binding to the receptor by immunofluorescence (IF) microscopy. Ex vivo experiments using both C57BL/6 wild type- and CXCR3−/− mice mounted in microsnapwell chambers were performed to measure zonulin release and intestinal transepithelial electrical resistance (TEER).

Results:

Three main proteins with estimated molecular weights of 93, 100, and 107 kDa, were eluted from the gliadin affinity column following adsorption of solubilized intestinal plasma membrane preparations. The bands were subjected to MALDI mass spec fingerprint analysis and the 100 kDa band was identified as the chemokine receptor CXCR3. In vitro IF experiments showed that gliadin bound on CXCR3-transfected cells but not on cells transfected with vector alone. The ex vivo experiments revealed that gliadin exposure to intestinal segments obtained from WT mice caused an increase zonulin release and a significant TEER decrement. Conversely, intestinal segments obtained from CXCR3 KO mice exposed to gliadin failed to release increased levels of zonulin and showed no TEER changes.

Conclusions:

Our data suggest that CXCR3 is a receptor targeted by gliadin in intestinal epithelial cells. The binding of gliadin to epithelial CXCR3 is followed by zonulin release and an increase in IP.

© 2006 Lippincott Williams & Wilkins, Inc.