Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3.

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 Alpha-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 alpha-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 alpha-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.

[1]  B. Powell,et al.  Rapid disruption of intestinal barrier function by gliadin involves altered expression of apical junctional proteins , 2005, FEBS letters.

[2]  Shizuo Akira,et al.  Toll-like receptor signalling , 2004, Nature Reviews Immunology.

[3]  N. Aiyar,et al.  Identification and pharmacological characterization of native, functional human urotensin‐II receptors in rhabdomyosarcoma cell lines , 2004, British journal of pharmacology.

[4]  Giuseppe Iacono,et al.  Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines , 2006, Scandinavian journal of gastroenterology.

[5]  Ronald H. Zielke,et al.  Role of the intestinal tight junction modulator zonulin in the pathogenesis of type I diabetes in BB diabetic-prone rats. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[6]  E. Kistner,et al.  Reprogramming of CTLs into natural killer–like cells in celiac disease , 2006, The Journal of experimental medicine.

[7]  H. R. Zielke,et al.  Affinity Purification and Partial Characterization of the Zonulin/Zonula Occludens Toxin (Zot) Receptor from Human Brain , 2000, Journal of neurochemistry.

[8]  V. Silano,et al.  Toxicity of Wheat Flour Proteins and Protein-Derived Peptides for In Vitro Developing Intestine from Rat Fetus , 1979, Pediatric Research.

[9]  M. Freedman,et al.  gamma/delta T cells in multiple sclerosis: chemokine and chemokine receptor expression. , 2002, Clinical immunology.

[10]  K. Kaukinen,et al.  Intraepithelial lymphocytes in celiac disease. , 2003 .

[11]  R. Strieter,et al.  Differential roles for CXCR3 in CD4+ and CD8+ T cell trafficking following viral infection of the CNS , 2006, European journal of immunology.

[12]  A. Ding,et al.  MyD88-mediated stabilization of interferon-γ-induced cytokine and chemokine mRNA , 2006, Nature Immunology.

[13]  H. Mollenkopf,et al.  Early granuloma formation after aerosol Mycobacterium tuberculosis infection is regulated by neutrophils via CXCR3‐signaling chemokines , 2003, European journal of immunology.

[14]  Andrew D. Luster,et al.  Intracellular Domains of CXCR3 That Mediate CXCL9, CXCL10, and CXCL11 Function* , 2004, Journal of Biological Chemistry.

[15]  A. Kraneveld,et al.  A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation , 2006, Nature Medicine.

[16]  D. Schuppan,et al.  Current concepts of celiac disease pathogenesis. , 2000, Gastroenterology.

[17]  S. Virgiliis,et al.  Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function , 2003, Gut.

[18]  L. Lanier,et al.  Evidence for NK Cell Subsets Based on Chemokine Receptor Expression1 , 2006, The Journal of Immunology.

[19]  W. Hancock,et al.  Requirement of the Chemokine Receptor CXCR3 for Acute Allograft Rejection , 2000, The Journal of experimental medicine.

[20]  S. Pereira,et al.  The pathogenesis of coeliac disease. , 2004, The international journal of biochemistry & cell biology.

[21]  R. D. Lynch,et al.  The tight junction: a multifunctional complex. , 2004, American journal of physiology. Cell physiology.

[22]  A. Luster,et al.  CXCR3 Requires Tyrosine Sulfation for Ligand Binding and a Second Extracellular Loop Arginine Residue for Ligand-Induced Chemotaxis , 2006, Molecular and Cellular Biology.

[23]  T. Junt,et al.  β cells are responsible for CXCR3-mediated T-cell infiltration in insulitis , 2002, Nature Medicine.

[24]  D. Schuppan,et al.  Identification of tissue transglutaminase as the autoantigen of celiac disease , 1997, Nature Medicine.

[25]  L. Adorini,et al.  Cutting Edge: Selective Usage of Chemokine Receptors by Plasmacytoid Dendritic Cells1 , 2001, The Journal of Immunology.

[26]  M. Barone,et al.  Growth factor-like activity of gliadin, an alimentary protein: implications for coeliac disease , 2006, Gut.

[27]  A. Fasano,et al.  Zonulin, a newly discovered modulator of intestinal permeability, and its expression in coeliac disease , 2000, The Lancet.

[28]  P. Matzinger,et al.  Danger signals: SOS to the immune system. , 2001, Current opinion in immunology.

[29]  Dario Iafusco,et al.  Zonulin Upregulation Is Associated With Increased Gut Permeability in Subjects With Type 1 Diabetes and Their Relatives , 2006, Diabetes.

[30]  A. Luster,et al.  Intracellular Domains of CXCR 3 That Mediate CXCL 9 , CXCL 10 , and CXCL 11 Function * , 2004 .

[31]  A. Luster,et al.  Chemokines--chemotactic cytokines that mediate inflammation. , 1998, The New England journal of medicine.

[32]  C. Wijmenga,et al.  Neutrophil recruitment and barrier impairment in celiac disease: a genomic study. , 2007, Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association.

[33]  H. Tse,et al.  Glutamate receptor ion channels : Activators and inhibitors , 2000 .

[34]  S. Rees,et al.  A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor. , 1997, Analytical biochemistry.

[35]  S. Romagnani.  Regulation of the T cell response , 2006, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[36]  T. Halstensen,et al.  Intraepithelial T Cells of the TcRγ/δ+CD8− and Vδ1/Jδ1+ Phenotypes are Increased in Coeliac Disease , 1989 .

[37]  T. Sørensen.  Targeting the chemokine receptor CXCR3 and its ligand CXCL10 in the central nervous system: potential therapy for inflammatory demyelinating disease? , 2004, Current neurovascular research.

[38]  M. Mayer,et al.  Glutamate receptor ion channels , 2005, Current Opinion in Neurobiology.

[39]  Sha Quan,et al.  CXCR3 Expression and Activation of Eosinophils: Role of IFN-γ-Inducible Protein-10 and Monokine Induced by IFN-γ1 , 2000, The Journal of Immunology.

[40]  M. Goldsmith,et al.  Multiple Extracellular Elements of CCR5 and HIV-1 Entry: Dissociation from Response to Chemokines , 1996, Science.

[41]  A. Fasano,et al.  Gliadin Stimulation of Murine Macrophage Inflammatory Gene Expression and Intestinal Permeability Are MyD88-Dependent: Role of the Innate Immune Response in Celiac Disease1 , 2006, The Journal of Immunology.

[42]  C. Wijmenga,et al.  Understanding the molecular basis of celiac disease: What genetic studies reveal , 2006, Annals of medicine.

[43]  G. Coppa,et al.  Host-dependent zonulin secretion causes the impairment of the small intestine barrier function after bacterial exposure. , 2002, Gastroenterology.

[44]  A. Fasano,et al.  Human zonulin, a potential modulator of intestinal tight junctions. , 2000, Journal of cell science.

[45]  L. Cosmi,et al.  CXCR3 and αEβ7 integrin identify a subset of CD8+ mature thymocytes that share phenotypic and functional properties with CD8+ gut intraepithelial lymphocytes , 2005, Gut.

[46]  K. Kaukinen,et al.  Intraepithelial Lymphocytes in Celiac Disease , 2003, American Journal of Gastroenterology.