Abstract
Celiac sprue is a T-cell-mediated enteropathy elicited in genetically susceptible individuals by dietary gluten proteins. To initiate and propagate inflammation, proteolytically resistant gluten peptides must be translocated across the small intestinal epithelium and presented to DQ2-restricted T cells, but the effectors enabling this translocation under normal and inflammatory conditions are not well understood. We demonstrate that a fluorescently labeled antigenic 33-mer gluten peptide is translocated intact across a T84 cultured epithelial cell monolayer and that preincubation of the monolayer with media from gluten-stimulated, celiac patient-derived intestinal T cells enhances the apical-to-basolateral flux of this peptide in a dose-dependent, saturable manner. The permeability-enhancing activity of activated T-cell media is inhibited by blocking antibodies against either interferon-γ or its receptor and is recapitulated using recombinant interferon-γ. At saturating levels of interferon-γ, activated T-cell media does not further increase transepithelial peptide flux, indicating the primacy of interferon-γ as an effector of increased epithelial permeability during inflammation. Reducing the assay temperature to 4°C reverses the effect of interferon-γ but does not reduce basal peptide flux occurring in the absence of interferon-γ, suggesting active transcellular transport of intact peptides is increased during inflammation. A panel of disease-relevant gluten peptides exhibited an inverse correlation between size and transepithelial flux but no apparent sequence constraints. Anti-interferon-γ therapy may mitigate the vicious cycle of gluten-induced interferon-γ secretion and interferon-γ-mediated enhancement of gluten peptide flux but is unlikely to prevent translocation of gluten peptides in the absence of inflammatory conditions.
Footnotes
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This work was supported in part by the National Institutes of Health [Grant T32-GM007276] (Cellular and Molecular Biology grant) (to M.T.B); the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01-DK063158] (to C.K.); and Achievement Rewards for College Scientists Foundation Predoctoral Scholarship (to M.S.).
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C.K. is a director and stockholder in Alvine Pharmaceuticals, a company that is developing a drug for celiac disease.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.148007.
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ABBREVIATIONS: GALT, gut-associated lymphoid tissue; TG2, transglutaminase 2 (or tissue transglutaminase); APC, antigen-presenting cell; IFN, interferon; TNF, tumor necrosis factor; MβCD, methyl β-cyclodextrin; FBS, fetal bovine serum; PEG, polyethylene glycol; RP-HPLC, reverse-phase high-performance liquid chromatography; LC-MS, liquid chromatography-assisted mass spectrometry; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; EP-B2, barley endoprotease B, isoform 2.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received October 27, 2008.
- Accepted February 12, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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