Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function.
OBJECTIVE
Despite the progress made in understanding the immunological aspects of the pathogenesis of coeliac disease (CD), the early steps that allow gliadin to cross the intestinal barrier are still largely unknown. The aim of this study was to establish whether gliadin activates a zonulin dependent enterocyte intracellular signalling pathway(s) leading to increased intestinal permeability.


METHODS
The effect of gliadin on the enterocyte actin cytoskeleton was studied on rat intestinal epithelial (IEC-6) cell cultures by fluorescence microscopy and spectrofluorimetry. Zonulin concentration was measured on cell culture supernatants by enzyme linked immunosorbent assay. Transepithelial intestinal resistance (Rt) was measured on ex vivo intestinal tissues mounted in Ussing chambers.


RESULTS
Incubation of cells with gliadin led to a reversible protein kinase C (PKC) mediated actin polymerisation temporarily coincident with zonulin release. A significant reduction in Rt was observed after gliadin addition on rabbit intestinal mucosa mounted in Ussing chambers. Pretreatment with the zonulin inhibitor FZI/0 abolished the gliadin induced actin polymerisation and Rt reduction but not zonulin release.


CONCLUSIONS
Gliadin induces zonulin release in intestinal epithelial cells in vitro. Activation of the zonulin pathway by PKC mediated cytoskeleton reorganisation and tight junction opening leads to a rapid increase in intestinal permeability.
Clemente
M G
MG
Department of Biomedical Sciences and Biotechnology, 2nd Pediatric Clinic, University of Cagliari, Cagliari, Italy.
De Virgiliis
S
S
Kang
J S
JS
Macatagney
R
R
Musu
M P
MP
Di Pierro
M R
MR
Drago
S
S
Congia
M
M
Fasano
A
A
D000199:Actins / Q000276:immunology; D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000032:analysis / Q000276:immunology*; D003513:Cycloheximide / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000276:immunology; D020895:Enterocytes / Q000187:drug effects* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005903:Gliadin / Q000276:immunology / Q000494:pharmacology*; D006242:Haptoglobins; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D011108:Polymers; D011493:Protein Kinase C / Q000378:metabolism; D011498:Protein Precursors; D011817:Rabbits; D051381:Rats; D015398:Signal Transduction / Q000276:immunology*; D013050:Spectrometry, Fluorescence / Q000379:methods
D000199:Actins; D006242:Haptoglobins; D011108:Polymers; D011498:Protein Precursors; C408043:zonulin; D005903:Gliadin; D002772:Cholera Toxin; D003513:Cycloheximide; D011493:Protein Kinase C