Gastroenterology

Gastroenterology

Volume 135, Issue 1, July 2008, Pages 194-204.e3
Gastroenterology

Basic—Alimentary Tract
Gliadin Induces an Increase in Intestinal Permeability and Zonulin Release by Binding to the Chemokine Receptor CXCR3

https://doi.org/10.1053/j.gastro.2008.03.023Get rights and content

Background & Aims: Celiac disease is an immune-mediated enteropathy triggered by gliadin, a component of the grain protein gluten. Gliadin induces an MyD88-dependent zonulin release that leads to increased intestinal permeability, a postulated early element in the pathogenesis of celiac disease. We aimed to establish the molecular basis of gliadin interaction with intestinal mucosa leading to intestinal barrier impairment. Methods: α-Gliadin affinity column was loaded with intestinal mucosal membrane lysates to identify the putative gliadin-binding moiety. In vitro experiments with chemokine receptor CXCR3 transfectants were performed to confirm binding of gliadin and/or 26 overlapping 20mer α-gliadin synthetic peptides to the receptor. CXCR3 protein and gene expression were studied in intestinal epithelial cell lines and human biopsy specimens. Gliadin-CXCR3 interaction was further analyzed by immunofluorescence microscopy, laser capture microscopy, real-time reverse-transcription polymerase chain reaction, and immunoprecipitation/Western blot analysis. Ex vivo experiments were performed using C57BL/6 wild-type and CXCR3−/− mouse small intestines to measure intestinal permeability and zonulin release. Results: Affinity column and colocalization experiments showed that gliadin binds to CXCR3 and that at least 2 α-gliadin 20mer synthetic peptides are involved in this binding. CXCR3 is expressed in mouse and human intestinal epithelia and lamina propria. Mucosal CXCR3 expression was elevated in active celiac disease but returned to baseline levels following implementation of a gluten-free diet. Gliadin induced physical association between CXCR3 and MyD88 in enterocytes. Gliadin increased zonulin release and intestinal permeability in wild-type but not CXCR3−/− mouse small intestine. Conclusions: Gliadin binds to CXCR3 and leads to MyD88-dependent zonulin release and increased intestinal permeability.

Section snippets

Reagents

Gliadin (crude wheat), pepsin, and trypsin were purchased from Sigma (St Louis, MO). Gliadin was pepsin/trypsin digested (PT-gliadin) as described previously10 with minor modifications.11 Recombinant α-gliadin was a gift from Dr D. Kasarda (USDA-ARS, Albany, CA). Recombinant interleukin (rIL)-1, monokine induced by interferon (IFN) γ (rMig/CXCL9), IFN-γ-inducible protein 10 (rIP-10/CXCL10), IFN-γ-inducible T-cell α-chemoattractant (rI-TAC/CXCL11), and tumor necrosis factor-α (rTNF-α) were

Identification of CXCR3 as the PT-Gliadin Intestinal Binding Protein

To identify the putative gliadin receptor, membrane fractions were prepared from rabbit small intestine and applied to an Affi-gel α-gliadin affinity column. Three main proteins with estimated molecular weights of 93, 100, and 107 kilodaltons were eluted from the affinity column and subjected to MALDI mass spectrometric fingerprint analysis following digestion with trypsin. The 100-kilodalton band was identified by mass spec/mass spec (MS/MS) as the chemokine receptor CXCR3, based on sequences

Discussion

TJs are central to the regulation of intestinal permeability because they maintain the contiguity of intestinal epithelial cells and are capable of prompt and coordinated responses to the many physiologic challenges to the intestinal epithelial barrier.4 Increased intestinal permeability appears to be an early biologic change that precedes the onset of autoimmune diseases, including CD and type I diabetes.8, 23, 24 The peculiarity of CD is that it is the only autoimmune disease for which the

References (44)

  • W. Dieterich et al.

    Identification of tissue transglutaminase as the autoantigen of celiac disease

    Nat Med

    (1997)
  • A.J. Monsuur et al.

    Understanding the molecular basis of celiac disease: what genetic studies reveal

    Ann Med

    (2006)
  • E.E. Schneeberger et al.

    The tight junction: a multifunctional complex

    Am J Physiol Cell Physiol

    (2004)
  • W. Wang et al.

    Human zonulin, a potential modulator of intestinal tight junctions

    J Cell Sci

    (2000)
  • S. Drago et al.

    Gliadin, zonulin and gut permeability: effects on celiac and non-celiac intestinal mucosa and intestinal cell lines

    Scand J Gastroenterol

    (2006)
  • M.G. Clemente et al.

    Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function

    Gut

    (2003)
  • G. De Ritis et al.

    Toxicity of wheat flour proteins and protein-derived peptides for in vitro developing intestine from rat fetus

    Pediatr Res

    (1979)
  • K.E. Thomas et al.

    Gliadin stimulation of murine macrophage inflammatory gene expression and intestinal permeability are MyD88-dependent: role of the innate immune response in celiac disease

    J Immunol

    (2006)
  • R. Lu et al.

    Affinity purification and partial characterization of the zonulin/zonula occludens toxin (Zot) receptor from human brain

    J Neurochem

    (2000)
  • R.A. Colvin et al.

    CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis

    Mol Cell Biol

    (2006)
  • M. Morimoto et al.

    Functional importance of regional differences in localized gene expression of receptors for IL-13 in murine gut

    J Immunol

    (2006)
  • S.A. Douglas et al.

    Identification and pharmacological characterization of native, functional human urotensin-II receptors in rhabdomyosarcoma cell lines

    Br J Pharmacol

    (2004)
  • Cited by (0)

    Supported in part by National Institutes of Health grants DK-48373 (to A.F.) and AI-18797 (to S.N.V.).

    Conflicts of interest: S.N.V. and A.F. have financial relationship with Alba Therapeutics.

    View full text