Gastroenterology

Gastroenterology

Volume 120, Issue 7, June 2001, Pages 1666-1679
Gastroenterology

Alimentary Tract
Colonic epithelial hPepT1 expression occurs in inflammatory bowel disease: Transport of bacterial peptides influences expression of MHC class 1 molecules*,**

https://doi.org/10.1053/gast.2001.24845Get rights and content

Abstract

Background & Aims: hPepT1 is an intestinal epithelial apical membrane transporter responsible for uptake of di/tripeptides (including bacterial derived proinflammatory n-formyl peptides). hPepT1 expression normally has a strict axial gradient—highest in the proximal small intestine with no expression in the colon. Methods: Small intestinal-like cells (Caco2-BBE), and colonic-like cells (HT29-Cl.19A), and colonic mucosa from diseased and control patients were used in the present study. Results: hPepT1 expression occurs aberrantly in the colon with chronic ulcerative colitis (6 patients) and Crohn's disease (4 patients), but not in normal colon (4 patients) or colon with microscopic colitis (4 patients). To model expression of hPepT1 by colonic-like cells in inflamed states, we stably transfected HT29-Cl.19A cells with a modified hPepT1 tagged on the N-terminus with green fluorescence protein. Analysis of transfected cells revealed that: GFP-hPepT1 protein, like the natural protein, is targeted to the apical plasma membrane. In addition, the tagged protein retains the capability of di/tripeptide absorption, and the expression of the tagged protein by HT29-Cl.19A cells permits absorption of N-formyl-methionyl-leucyl-phenylalanine (fMLP), as occurs in hPepT1 expressing Caco2-BBE cells. fMLP uptake by colonic cells expressing GFP-hPepT1 specifically enhances major histocompatibility complex class I surface expression. Conclusions: These data collectively indicate that, in some states of chronic inflammation, hPepT1 may be anomolously expressed in the colon. Further, transport of fMLP by hPepT1 potentially stimulates expression of key accessory immune molecule, MHC-1.

GASTROENTEROLOGY 2001;120:1666-1679

Section snippets

Cell culture

Caco2-BBE,31 HT29-Cl.19A32, 33 were grown as confluent monolayers in a 1:1 mixture of Dulbecco's Vogt modified Eagle's media and Ham's F-12 medium supplemented with 15 mmol/L HEPES buffer (pH 7.5), 14 mmol/L NaHCO3, and 10% new-born calf serum. Monolayers were subcultured every 7 days by trypsinization with 0.1% trypsin and 0.9 mmol/L ethylenediaminetetraacetic acid (EDTA) in Ca2+/Mg2+-free phosphate-buffered saline (PBS). Transfected cell lines were maintained in the same media containing 1.2

hPepT1 is expressed in normal human small intestine but not in normal colon

As shown by immunostaining in Figure 1A, enterocytes of normal human ileum display apical hPepT1 (these experiments were repeated on 4 different tissue sets, and similar results were obtained).

. hPepT1 protein is expressed in normal human ileum, but not in normal human colon. Frozen sections were stained with anti-hPepT1 antibody. Sections were counterstained for nuclei with methyl green. (A) In human ileum (normal ileal mucosa, male 56 years old), hPepT1 is localized to the brush border (arrow).

Discussion

Here we show that a transporter, which we have previously showed to be able to transport the bacterial product fMLP and alter subsequent epithelial-neutrophil interactions,21 can be aberrantly expressed in the colon under chronic inflammation. A tagged fusion chimera of this transporter, hPepT1, was made and shown to be functional and surface expressed, and was used to transfect a hPepT1 negative colonic-like epithelial model to perform further in vitro analysis of the above in vivo

Acknowledgements

The authors thank Dr. P. E. Jensen (Emory University, Atlanta, GA) for his advice and are grateful to Dr. T. Ziegler (Emory University) for providing human biopsy samples.

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    *

    Address requests for reprints to: Didier Merlin, Ph.D., Emory University, Department of Pathology and Laboratory Medicine, 1639 Pierce Drive, Atlanta, Georgia 30322. e-mail: [email protected]; fax: (419) 821-3041.

    **

    Supported by National Institutes of Health grants DK-47662 and DK-35932 (to J.L.M.), AR44268 (to I.W.), and DK-4317 (to M.A.H.). This work was initiated with a National Research Service Award DK 09800 (to D.M.). D. Merlin and S.V. Sitaraman are currently recipients of a Career Development Award (Crohn's & Colitis Foundation of America).

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