Volume 120, Issue 7, Pages 1666-1679 (June 2001) Colonic epithelial hPepT1 expression occurs in inflammatory bowel disease: Transport of bacterial peptides influences expression of MHC class 1 molecules  Didier Merlin, Mustapha Si-Tahar, Shanthi V. Sitaraman, Karen Eastburn, Ifor Williams, Xia Liu, Matthias A. Hediger, James L. Madara  Gastroenterology  Volume 120, Issue 7, Pages 1666-1679 (June 2001) DOI: 10.1053/gast.2001.24845 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 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). (C) In normal colon (normal colonic mucosa, male 56 years old), no apical staining of hPepT1 is observed. Total membrane proteins (50 μg/lane) from ileum (B1 and B2) or colonic (D) mucosal biopsy samples were subjected to 10% SDS-PAGE followed by transfer to nitrocellulose membrane. The blot was immunostained with anti-hPepT1 antibody in the absence (A, B2, C, D) or in presence (B1) of 10 μmol/L peptide antigen. Gastroenterology 2001 120, 1666-1679DOI: (10.1053/gast.2001.24845) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Chronic colitis is associated with hPepT1 expression in the human colon. (A) Samples obtained from colonic mucosa lanes 1 and 2: (chronic ulcerative colitis; colon; female 33 years old), lanes 3 and 4: (normal colon and colon; female 42 years old). Tissues were subjected to RT-PCR using 2 sets of hPepT1-specific primers as described in Methods. The RT-PCR products were analyzed by agarose gel electrophoresis. The expected size of the products were: PT2, 1 kb (lane 1); and PT1, 1.6 kb (lane 2). Normal colon did not express any detectable product (lanes 3 and 4), whereas chronic ulcerative colitis expressed the products PT2 and PT1 (lanes 1 and 2). β actin specific primers were used as controls. (B) Frozen sections were stained as described in Figure 1. Frozen sections of mucosa of chronic ulcerative colitis (chronic active colitis; colon; female 22 years old; B) and Crohn's disease (colon; male 42 years old; D) were stained with anti-hPepT1 (D). As controls, mucosa of chronic ulcerative colitis (chronic active colitis; colon; female 22 years old) was stained with preimmune sera (C). Specific apical staining for hPepT1 was observed in colonic cells with ulcerative chronic colitis and Crohn's disease (arrow). Gastroenterology 2001 120, 1666-1679DOI: (10.1053/gast.2001.24845) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 hPepT1 is apically expressed in Caco2-BBE monolayers (small intestine cell-like), but not expressed in HT29-Cl.19A (colonic cell-like). (A) Northern blot analysis was performed on total RNA (20 μg) from HT29-Cl.19A cells (lane 1) or from Caco2-BBE cells (lane 2). A 3.3-kb hybridizing signal was present in Caco2-BBE, but not in HT29-Cl.19A. The same blot was stripped and reprobed with the β-actin cDNA. Filter-grown Caco2-BBE monolayers (B) and HT29-Cl.19A (D) were fixed, permeabilized, and stained with anti-hPepT1 followed by appropriate fluorescein isothiocyanate–conjugated secondary antibodies. Filter-grown monolayers were subjected to domain-specific biotinylation (Ap, apical domain; Bl, basolateral domain) for 30 minutes followed by Western blot analysis of total cell lysate from Caco2-BBE (C) or HT29-Cl.19A cells (E). Total cell protein was subjected to 10% SDS-PAGE followed by transfer to nitrocellulose membrane. The blot was immunostained with anti-hPepT1 antibody. Gastroenterology 2001 120, 1666-1679DOI: (10.1053/gast.2001.24845) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Caco2-BBE but not HT29-Cl.19A monolayers transport Glycylsarcosine. Caco2-BBE or HT29-Cl.19A monolayers were incubated apically for 60 minutes at 37°C at pH 6.2 with 10 μmol/L [14C] Glycylsarcosine (Gly-Sar) with and without 10 mmol/L Gly-Leu. Thereafter, medium was aspirated, and cells were rapidly washed twice with 2 mL ice-cold incubation media. Cells were solubilized in 1 mL of 1N NaOH, and radioactivity was determined by liquid scintillation counting. Each column represents mean ± SD of 4 monolayers. Gastroenterology 2001 120, 1666-1679DOI: (10.1053/gast.2001.24845) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Apical domain-specific plasma membrane delivery of GFP-hPepT1 in stably transfected colonic cells (HT29-Cl.19A). (A) Analysis of transfectants of HT29-Cl.19A cells by flow cytometry: untransfected HT29-Cl.19A cells (red histogram) or those transfected with GFP fused to hPepT1 (open histogram). (B) Total membrane protein (50 μg/lane) from HT29-Cl.19A transfected with the native hPepT1 (lane 1), with the pCB6 vector only (lane 2), with the pEGFP-C3 vector, and with GFP-hPepT1 (lane 4). Samples were subjected to 10% SDS-PAGE followed by transfer to nitrocellulose membranes. The blots were immunostained with anti-hPepT1 antibody. (C) Confocal microscopic localization of GFP-hPepT1 (green) in HT29-Cl.19A-GFP-hPepT1 monolayers also stained with rhodamine phalloidin (actin, red). En face views (at level of apical domain (Ap) or basolateral domain (Bl). (D) Filter-grown HT29-Cl.19A-GFP-hPepT1 monolayers were subjected to domain-specific biotinylation (Ap, apical domain; Bl, basolateral domain) for 30 minutes followed by Western Blot analysis of total cells from HT29-Cl.19A GFP-hPepT1. Total cell protein was subjected to 10% SDS-PAGE followed by transfer to nitrocellulose membrane. The blot was immunostained with anti-hPepT1 antibody. Gastroenterology 2001 120, 1666-1679DOI: (10.1053/gast.2001.24845) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 HT29-Cl.19A GFP-hPepT1 monolayers transport dipeptides and fMLP. HT29-Cl.19A monolayers transfected with GFP alone or GFP-hPepT1 were: (A) incubated for 60 minutes at 37°C at pH 6.2 with 10 μmol/L [14C] Glycylsarcosine (Gly-Sar) with or without 10 mmol/L Gly-Leu; (B) incubated with 100 nmol/L [3H] fMLP for 110 minutes at 37°C at pH 6.2 with or without 10 mmol/L Gly-Leu. Thereafter, media was aspirated, and cells were rapidly washed twice with 2 mL ice-cold incubation medium. Cells were solubilized in 1 mL of 1N NaOH, and radioactivity was determined by liquid scintillation. (Inset A) Filter grown HT29-Cl.19A-GFP cells or HT29.Cl.19A-GFP-hPepT1 cells were apically incubated for 1 hour with 20 mmol/L β Ala-Lys-Nϵ-AMCA. After incubation, the cells were rinsed 3 times with ice-cold HBSS. After fixation with 3.7% paraformaldehyde in PBS, filters were mounted on slides and viewed under fluorescence microscope equipped with the appropriate filter combinations. Each column represents mean ± of 5 filters. *P < 0.05 HT29-Cl.19A-GFP vs. HT29-Cl.19A-GFP-hPepT1. Gastroenterology 2001 120, 1666-1679DOI: (10.1053/gast.2001.24845) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 fMLP uptake by HT29-Cl.19A-GFP-hPepT1 induces MHC Class I expression. Flow cytometric analysis of MHC Class I on HT29-Cl.19A-GFP-hPepT1 cells (A) or HT29-Cl.19A-GFP cells (B). Monolayers were untreated or treated with interferon γ (1000 U/mL for 30 hours), fMLP (50 μmol/L for 30 hours), Gly-Leu (50 μmol/L for 30 hours). Subsequently, cells were harvested and stained with MHC Class I (W6/32) antibody. Cells were analyzed by FACS (traces in black, cells without antibody; in green, cells with W6/32 antibody; in red, cells treated with fMLP in presence of W6/32 antibody; in blue, cells treated with interferon γ in presence of W6/32 antibody). In (C), data are expressed as fold increase over the baseline value calculated by dividing the mean fluorescence intensity (of treated cells) by mean fluorescence intensity (of untreated cells). Each bar is representative of results from 5 to 15 different monolayers for each condition (HT29-Cl.19A GFP-hPepT1: Gly-Leu, n = 7; Interferon γ [IFN γ], n = 6; fMLP, n = 15; HT29-Cl.19A GFP: Gly-Leu, n = 5; IFN γ, n = 6; fMLP, n = 10; where n represents the number of monolayers tested). *P < 0.05 HT29-Cl.19A GFP cells treated with fMLP vs. HT29-Cl.19A GFP-hPepT1 treated with fMLP. Gastroenterology 2001 120, 1666-1679DOI: (10.1053/gast.2001.24845) Copyright © 2001 American Gastroenterological Association Terms and Conditions