Volume 134, Issue 1, Pages 179-191 (January 2008) A Prominent Role for Mucosal Cystine/Cysteine Metabolism in Intestinal Immunoregulation  Bernd Sido, Felix Lasitschka, Thomas Giese, Nikolaus Gassler, Benjamin Funke, Jutta Schröder–Braunstein, Ulf Brunnemer, Stefan C. Meuer, Frank Autschbach  Gastroenterology  Volume 134, Issue 1, Pages 179-191 (January 2008) DOI: 10.1053/j.gastro.2007.11.001 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Total glutathione content in LP-T from normal gut mucosa vs autologous PB-T. Data are presented as means ± SD of 8 independent experiments. Gastroenterology 2008 134, 179-191DOI: (10.1053/j.gastro.2007.11.001) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 (A) Cysteine concentrations in the supernatants of LP-MO from normal gut vs autologous PB-MO after 40 hours of culture. Data represent means ± SD of 6 independent experiments. (B) Transmembrane transport activity for L-[35S]cystine (50 μmol/L) via system xc− in lamina propria cells from normal gut vs autologous peripheral blood cells. Specificity of system xc− was confirmed through inhibition of cystine uptake by L-glutamate. Results are presented as means ± SD of 6 independent experiments. (C) Representative Western blot analysis of xCT and β-actin in PB-MO vs LP-MO from normal gut mucosa. (D) Quantitative xCT and 4F2hc mRNA expression in LP-MO from normal gut vs autologous PB-MO. Values represent means ± SD of 3 independent experiments. Gastroenterology 2008 134, 179-191DOI: (10.1053/j.gastro.2007.11.001) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 In situ expression of the cystine transporter subunit xCT. (A) Immunoenzyme staining of xCT in normal colonic mucosa (NC) and inflamed colonic mucosa in UC and CD; NegC, negative control; original magnifications, ×30 for NC and NegC and ×60 for UC and CD. (B) Immunofluorescence staining of xCT in the normal colonic mucosa (NC) and inflamed ileum in CD. When compared with corresponding negative controls (NegC), some intestinal epithelia display weak positive stainings at their apical membrane (arrow). Gastroenterology 2008 134, 179-191DOI: (10.1053/j.gastro.2007.11.001) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Phenotypic characterization of xCT+ cells in situ in IBD. Double immunofluorescence staining of xCT, red signals, and various cellular subtype-specific antigens, green signals, in the inflamed ileum in CD. Colocalization of both antigens is shown by yellow signals in the overlay (OL). Gastroenterology 2008 134, 179-191DOI: (10.1053/j.gastro.2007.11.001) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 (A) Quantitative expression of xCT mRNA in transmural gut samples (n = 101) of patients with UC and CD and normal controls. Transcript levels of xCT are significantly increased only in inflamed tissue in UC (n = 24) and CD (n = 29; 17 ileum, 12 colon) but not in noninflamed tissues in UC (n = 13) or CD (n = 18; 8 ileum, 10 colon) as compared with normal controls (n = 17; 9 colon, 8 ileum). Box plots (median, 25% and 75% percentile, minimum and maximum); *P ≤ .001 vs control. (B) Western blot analysis of xCT in mucosal isolates from patients with UC and CD and normal controls. Bracket indicates 2 samples derived from the same CD patient. (C) Cysteine secretion by LP-MO from inflamed colon in UC vs autologous PB-MO after 40 hours of culture with or without lipopolysaccharide (1 μg/mL). Data represent means ± SD of 6 independent experiments. (D) Thiol-dependent proliferation of isolated LP-T in UC following stimulation via CD3. Cultures were primarily set up in cystine-deficient medium and supplemented with either cysteine (30 μmol/L) or equimolar amounts of cystine (15 μmol/L) as described in the Materials and Methods section. Results are expressed as mean counts per minute of triplicate cultures ± SD and are representative of 3 independent experiments. Gastroenterology 2008 134, 179-191DOI: (10.1053/j.gastro.2007.11.001) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 (A) GSH staining of lamina propria cells in normal colonic mucosa (NC) and IBD. NegC, negative control. Alkaline phosphatase; magnifications, ×60. (B) Quantitative analysis of GSH expression in situ in IBD vs normal controls (Con) (total number, n = 57). The percentage of GSH-positive mucosal cells is significantly increased in inflamed areas in UC (n = 10) as well as colonic (n = 15) and ileal (n = 7) CD as compared with the respective colonic (n = 14) and ileal (n = 11) control samples. Box plots (median, 25% and 75% percentile, minimum and maximum); *P < .05, **P < .001 vs control. (C) Double immunofluorescence staining for GSH and CD3 in the inflamed colonic mucosa in UC. Most CD3+ cells stain positive for GSH, yellow signal in the overlay (OL). Gastroenterology 2008 134, 179-191DOI: (10.1053/j.gastro.2007.11.001) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 GSH levels in CD3+ LP-T from inflamed mucosa in IBD as compared with CD3+ LP-T from normal gut. LP-T and autologous PB-T isolated from a patient with active UC (A and B), or from a control patient (C and D), were double stained for CD3 and intracellular GSH and analyzed by flow cytometry. A representative example of GSH staining is shown as histograms. Data depict the percentage of CD3+ T cells being positive for GSH and the mean fluorescence intensity (MFI) of GSH staining. NegC, negative control. Gastroenterology 2008 134, 179-191DOI: (10.1053/j.gastro.2007.11.001) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 Quantitative gene expression analysis of the catalytic heavy subunit of γGCS in transmural gut samples (n = 101) of patients with UC and CD and normal controls. Transcript levels of γGCS are significantly decreased in inflamed tissue in UC (n = 24) and CD (n = 29; 17 ileum, 12 colon) but not in noninflamed tissue in UC (n = 13) or CD (n = 18; 8 ileum, 10 colon) as compared with normal controls (n = 17; 9 colon, 8 ileum). Box plots (median, 25% and 75% percentile, minimum and maximum); *P < .02, **P = .001 vs control. Gastroenterology 2008 134, 179-191DOI: (10.1053/j.gastro.2007.11.001) Copyright © 2008 AGA Institute Terms and Conditions