A Prominent Role for Mucosal Cystine/Cysteine Metabolism in Intestinal Immunoregulation
2007; Elsevier BV; Volume: 134; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2007.11.001
ISSN1528-0012
AutoresBernd Sido, Felix Lasitschka, Thomas Giese, Nikolaus Gaßler, Benjamin Funke, Jutta Schröder–Braunstein, Ulf Brunnemer, Stefan Meuer, Frank Autschbach,
Tópico(s)Immune cells in cancer
ResumoBackground & Aims: T-cell receptor reactivity of intestinal lamina propria T cells (LP-T) critically depends on the capacity of local accessory cells to secrete cysteine. For T cells, cysteine is the limiting precursor for glutathione synthesis, a prerequisite for antigen-dependent proliferation. We aimed to determine the role of the redoxactive microenvironment for hyporeactivity of LP-T in normal human gut vs hyperreactivity of LP-T in inflammatory bowel disease. Methods: Parameters relevant to cysteine production, determined as acid-soluble thiol, by intestinal lamina propria macrophages (LP-MO) vs peripheral blood monocytes were investigated (L-[35S]cystine uptake via system xc−, messenger RNA, and protein expression of the cystine transporter subunit xCT). Glutathione levels in LP-T and peripheral blood T cells were analyzed both spectrophotometrically and by immunofluorescent staining in situ and in vitro. Results: LP-MO from normal gut, unlike peripheral blood monocytes, are unable to take up cystine, which is due to a deficient expression of the transporter xCT in situ and in vitro. As a consequence, LP-MO do not secrete cysteine. The glutathione content in LP-T from normal gut is <50% of that in autologous peripheral blood T cells. In contrast, in inflammatory bowel disease, CD14+CD68+ LP-MO express xCT and secrete substantial amounts of cysteine upon stimulation, which results in high glutathione levels and full T-cell receptor reactivity in LP-T. Conclusions: The antioxidative microenvironment of LP-T in inflammatory bowel disease and the prooxidative microenvironment in normal gut explain the differential T-cell receptor reactivities. Background & Aims: T-cell receptor reactivity of intestinal lamina propria T cells (LP-T) critically depends on the capacity of local accessory cells to secrete cysteine. For T cells, cysteine is the limiting precursor for glutathione synthesis, a prerequisite for antigen-dependent proliferation. We aimed to determine the role of the redoxactive microenvironment for hyporeactivity of LP-T in normal human gut vs hyperreactivity of LP-T in inflammatory bowel disease. Methods: Parameters relevant to cysteine production, determined as acid-soluble thiol, by intestinal lamina propria macrophages (LP-MO) vs peripheral blood monocytes were investigated (L-[35S]cystine uptake via system xc−, messenger RNA, and protein expression of the cystine transporter subunit xCT). Glutathione levels in LP-T and peripheral blood T cells were analyzed both spectrophotometrically and by immunofluorescent staining in situ and in vitro. Results: LP-MO from normal gut, unlike peripheral blood monocytes, are unable to take up cystine, which is due to a deficient expression of the transporter xCT in situ and in vitro. As a consequence, LP-MO do not secrete cysteine. The glutathione content in LP-T from normal gut is <50% of that in autologous peripheral blood T cells. In contrast, in inflammatory bowel disease, CD14+CD68+ LP-MO express xCT and secrete substantial amounts of cysteine upon stimulation, which results in high glutathione levels and full T-cell receptor reactivity in LP-T. Conclusions: The antioxidative microenvironment of LP-T in inflammatory bowel disease and the prooxidative microenvironment in normal gut explain the differential T-cell receptor reactivities. The intestinal mucosal surface is in permanent contact with a multitude of foreign antigens of bacterial and nutritional origin. However, although the gut harbors the largest T-cell compartment of the body, mucosal T cells within the lamina propria (LP-T) normally do not produce systemic adaptive T-cell responses against luminal components. In inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD), this natural unresponsiveness is lost.1Jump R.L. Levine A.D. Mechanisms of natural tolerance in the intestine.Inflamm Bowel Dis. 2004; 10: 462-478Google Scholar IBD is characterized by relative T-cell receptor (TCR) hyperreactivity of LP-T with proliferation rates upon TCR stimulation that are comparable with those of peripheral blood T cells (PB-T),2Pirzer U. Schönhaar A. Fleischer B. et al.Reactivity of infiltrating T lymphocytes with microbial antigens in Crohn's disease.Lancet. 1991; 338: 1238-1239Google Scholar, 3Qiao L. Golling M. Autschbach F. et al.T-cell receptor repertoire and mitotic responses of lamina propria T lymphocytes in inflammatory bowel disease.Clin Exp Immunol. 1994; 97: 303-308Google Scholar accompanied by immune responses against luminal antigens.4Duchmann R. Kaiser I. Hermann E. et al.Tolerance exists towards resident intestinal microflora but is broken in active inflammatory bowel disease.Clin Exp Immunol. 1995; 102: 448-455Google Scholar, 5Macpherson A. Khoo U.Y. Forgacs I. et al.Mucosal antibodies in inflammatory bowel disease are directed against intestinal bacteria.Gut. 1996; 38: 365-375Google Scholar Moreover, inflamed mucosa contains large numbers of activated macrophages.6Rugtveit J. Brandtzaeg P. Halstensen T.S. et al.Increased macrophage subset in inflammatory bowel disease: apparent recruitment from peripheral blood monocytes.Gut. 1994; 35: 669-674Google Scholar, 7Burgio V.L. Fais S. Boirivant M. et al.Peripheral monocyte and naive T-cell recruitment and activation in Crohn's disease.Gastroenterology. 1995; 109: 1029-1038Google Scholar, 8Grimm M.C. Pullman W.E. Bennett G.M. et al.Direct evidence of monocyte recruitment to inflammatory bowel disease mucosa.J Gastroenterol Hepatol. 1995; 10: 387-395Google Scholar, 9Mahida Y.R. The key role of macrophages in the immunopathogenesis of inflammatory bowel disease.Inflamm Bowel Dis. 2000; 6: 21-33Google Scholar The TCR reactivity of LP-T is, at least in part, delicately controlled by oxidoreductive processes, the mainstay of which exists in the availability of cysteine in the mucosal microenvironment.10Sido B. Braunstein J. Breitkreutz R. et al.Thiol-mediated redox regulation of intestinal lamina propria T-lymphocytes.J Exp Med. 2000; 192: 907-912Google Scholar For T lymphocytes, cysteine is the limiting precursor for the synthesis of the tripeptide glutathione,11Ishii T. Sugita Y. Bannai S. Regulation of glutathione levels in mouse spleen lymhocytes by transport of cysteine.J Cell Physiol. 1987; 133: 330-336Google Scholar the most abundant intracellular thiol and antioxidant. Sufficient amounts of glutathione are a prerequisite for cell cycle progression and lymphocyte proliferation following TCR stimulation: a 10%–30% decrease in the intracellular glutathione concentration of PB-T has been found to completely abolish TCR-stimulated Ca2+ signaling.12Staal F.J.T. Anderson M.T. Staal G.E. et al.Redox regulation of signal transduction: tyrosine phosphorylation and calcium influx.Proc Natl Acad Sci U S A. 1994; 91: 3619-3622Google Scholar T lymphocytes are physiologically unable to produce cysteine through intracellular reduction of the oxidized derivative cystine because their transmembrane transport activity for cystine is constitutively low.11Ishii T. Sugita Y. Bannai S. Regulation of glutathione levels in mouse spleen lymhocytes by transport of cysteine.J Cell Physiol. 1987; 133: 330-336Google Scholar, 13Gmünder H. Eck H.P. Dröge W. Low membrane transport activity for cystine in resting and mitogenically stimulated human lymphocyte preparations and human T cell clones.Eur J Biochem. 1991; 201: 113-117Google Scholar In contrast, cysteine can be easily taken up by T cells.11Ishii T. Sugita Y. Bannai S. Regulation of glutathione levels in mouse spleen lymhocytes by transport of cysteine.J Cell Physiol. 1987; 133: 330-336Google Scholar, 13Gmünder H. Eck H.P. Dröge W. Low membrane transport activity for cystine in resting and mitogenically stimulated human lymphocyte preparations and human T cell clones.Eur J Biochem. 1991; 201: 113-117Google Scholar This is, however, of only limited value because cysteine concentrations in body fluids are extremely low.14Mansoor M.A. Svardal A.M. Ueland P.M. Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma.Anal Biochem. 1992; 200: 218-229Google Scholar Therefore, TCR reactivity is strictly dependent on the ability of local accessory cells to secrete cysteine, the latter being taken up by T cells and employed for their glutathione synthesis.10Sido B. Braunstein J. Breitkreutz R. et al.Thiol-mediated redox regulation of intestinal lamina propria T-lymphocytes.J Exp Med. 2000; 192: 907-912Google Scholar, 15Gmünder H. Eck H.P. Benninghoff B. et al.Macrophages regulate intracellular glutathione levels of lymphocytes Evidence for an immunoregulatory role of cysteine.Cell Immunol. 1990; 129: 32-46Google Scholar With respect to the intestinal immune system, we have previously shown that resident lamina propria macrophages (LP-MO) isolated from the normal gut are unable to produce cysteine in vitro, whereas autologous peripheral blood monocytes (PB-MO) constitutively secrete cysteine, the amount of which is considerably enhanced upon stimulation by lipopolysaccharide or, alternatively, cross-linking of the CD58 receptor.10Sido B. Braunstein J. Breitkreutz R. et al.Thiol-mediated redox regulation of intestinal lamina propria T-lymphocytes.J Exp Med. 2000; 192: 907-912Google Scholar PB-MO avidly take up cystine via the Na+-independent anionic amino acid transport system xc− and secrete substantial amounts of cysteine following intracellular reduction of the disulfide.16Eck H.P. Dröge W. Influence of the extracellular glutamate concentration on the intracellular cyst(e)ine concentration in macrophages and on the capacity to release cysteine.Biol Chem Hoppe-Seyler. 1989; 370: 109-113Google Scholar Consequently, PB-MO can fully restore the TCR reactivity of LP-T from normal gut in coculture experiments, whereas resident LP-MO do not.10Sido B. Braunstein J. Breitkreutz R. et al.Thiol-mediated redox regulation of intestinal lamina propria T-lymphocytes.J Exp Med. 2000; 192: 907-912Google Scholar Importantly, addition of cysteine to LP-T is able to completely substitute for the costimulatory activity of PB-MO in vitro.10Sido B. Braunstein J. Breitkreutz R. et al.Thiol-mediated redox regulation of intestinal lamina propria T-lymphocytes.J Exp Med. 2000; 192: 907-912Google Scholar, 17Sido B. Breitkreutz R. Seel C. et al.Redox processes regulate intestinal lamina propria T lymphocytes.Methods Enzymol. 2002; 352: 232-247Google Scholar In the present study, we have investigated the question of whether the low TCR reactivity of LP-T in the physiologic cysteine-deficient mucosal microenvironment is associated with low intracellular glutathione concentrations. Moreover, we addressed the possibility that a sustained recruitment of PB-MO to the inflamed tissue in IBD6Rugtveit J. Brandtzaeg P. Halstensen T.S. et al.Increased macrophage subset in inflammatory bowel disease: apparent recruitment from peripheral blood monocytes.Gut. 1994; 35: 669-674Google Scholar, 7Burgio V.L. Fais S. Boirivant M. et al.Peripheral monocyte and naive T-cell recruitment and activation in Crohn's disease.Gastroenterology. 1995; 109: 1029-1038Google Scholar, 8Grimm M.C. Pullman W.E. Bennett G.M. et al.Direct evidence of monocyte recruitment to inflammatory bowel disease mucosa.J Gastroenterol Hepatol. 1995; 10: 387-395Google Scholar enhances the availability of cysteine in the microenvironment of LP-T and, thus, would increase their glutathione levels, restore their TCR reactivity, and lead to antigen-driven T-cell proliferation with the result of intestinal inflammation. Intestinal tissue samples from 47 patients with UC and 66 patients with CD were included in the present studies. Diagnosis was established by clinical and conventional histopathologic criteria. Histologically normal control tissues were derived from 48 patients with localized colon cancer or nonmalignant disorders. Patient details are provided in Supplementary Table 1 see (Supplementary Table 1 online at www.gastrojournal.org). LP-T and LP-MO were isolated from gut mucosa of fresh surgical specimens essentially as described earlier with the exception that LP-MO were also purified by anti-CD33 monoclonal antibody (mAb)-coated magnetic MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany).17Sido B. Breitkreutz R. Seel C. et al.Redox processes regulate intestinal lamina propria T lymphocytes.Methods Enzymol. 2002; 352: 232-247Google Scholar, 18Qiao L. Braunstein J. Golling M. et al.Differential regulation of human T-cell responsiveness by mucosal vs blood monocytes.Eur J Immunol. 1996; 26: 922-927Google Scholar LP-T were 90% positive for CD3. For comparison of lamina propria cells with peripheral blood cells, PB-T and PB-MO were obtained from the same patient and simultaneously processed. All cells were finally resuspended in RPMI 1640 plus 10% fetal calf serum (FCS), penicillin, streptomycin, and 2% glutamine (Sigma, Munich, Germany). Written informed consent was obtained from the patients. All studies were approved by the Ethical Committee of the University of Heidelberg and were conducted according to the principles expressed in the Helsinki Declaration. Purified T cells were allowed to rest for at least 12 hours at 37°C in complete medium prior to analysis. Cells (8 × 106) were lyzed in 200 μL sulfosalicylic acid (2.5%), incubated for 10 minutes on ice, and centrifuged. The supernatant was stored at −80°C until analysis. Total glutathione was determined spectrophotometrically (Victor; Wallac, Freiburg, Germany) using 96-well ELISA plates as described previously.17Sido B. Breitkreutz R. Seel C. et al.Redox processes regulate intestinal lamina propria T lymphocytes.Methods Enzymol. 2002; 352: 232-247Google Scholar The method is based on the catalytic action of reduced glutathione (GSH) in the reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by glutathione reductase in a cycling NADPH-dependent reaction. Cells (5 × 105/mL) were plated 1-mL volumes in 48-well culture plates in the absence or presence of 1 μg/mL lipopolysaccharide (from Escherichia coli, serotype 055:B5; Sigma). Cysteine was determined spectrophotometrically as acid-soluble thiol in cell-free supernatants after 40 hours of culture following derivatization with 5,5'-dithiobis(2-nitrobenzoic acid), essentially as described.10Sido B. Braunstein J. Breitkreutz R. et al.Thiol-mediated redox regulation of intestinal lamina propria T-lymphocytes.J Exp Med. 2000; 192: 907-912Google Scholar As identified by high-performance liquid chromatography, cysteine is the sole acid-soluble thiol present in tissue culture supernatants of PB-MO under these experimental conditions.17Sido B. Breitkreutz R. Seel C. et al.Redox processes regulate intestinal lamina propria T lymphocytes.Methods Enzymol. 2002; 352: 232-247Google Scholar In separate experiments, a blocking CD14 mAb (IgG1; R&D Systems, Wiesbaden, Germany) was added at 25 μg/mL to inhibit lipopolysaccharide-induced (0.1 μg/mL) cysteine secretion. Concentration-matched mouse IgG1 (R&D Systems) was used for negative controls. Cells were washed twice in sodium-free uptake medium (137 mmol/L choline chloride, 10 mmol/L HEPES, 5 mmol/L glucose, 0.7 mmol/L K2HPO4, 1 mmol/L calcium chloride, 1 mmol/L magnesium chloride; pH 7.4) and resuspended at 107 cells/mL in uptake medium. Following equilibration for 10 minutes at 37°C, 100 μL of the cell suspension was layered on top of 20 μL urea (6 mol/L) and 150 μL oil mixture (dibutylphtalate and dinonylphtalate 9:1, vol/vol). Eighty microliters of prewarmed L-[35S]cystine (Amersham, Freiburg, Germany) in uptake medium was added to the cell suspension at 50 μmol/L final concentration (final activity, 1–2 μCi/mL). After incubation for 0 minutes and 2 minutes at 37°C, cystine uptake was terminated by separating the cells from the uptake medium by centrifugation (8000g, 1 minute) and freezing the tube at −20°C for 1 hour. The tip of the tube was then cut above the urea layer and transferred to 3 mL scintillation fluid (Ultima Gold LSC Cocktail; Packard, Meriden, CT), and radioactivity was measured in a LS 1701 Liquid Scintillation Counter (Beckman Coulter, Fullerton, CA). Background activity (0 minutes) was substracted from the value obtained after 2 minutes of incubation. Cystine uptake was determined under conditions approaching initial rates of uptake, which increased linearly during the 2-minute incubation interval. Experiments were set up in duplicates. For stimulation of cells via CD3, 24-well plates (Costar, Bodenheim, Germany) were incubated overnight at 4°C with goat anti-mouse IgG+IgM, 1/250 in phosphate-buffered saline (PBS) (Dianova, Hamburg, Germany), washed 3 times, and then precoated with OKT3 mAb (1 μg/mL in PBS/1% bovine serum albumin) overnight at 4°C. LP-T were plated at 8 × 105/well in 1 mL cystine-deficient RPMI 1640/10% FCS (Cambrex, Verviers, Belgium). Cultures were supplemented with either cysteine (30 μmol/L) or equimolar amounts of cystine (15 μmol/L = 30 μmol/L cysteine equivalents) in 15-μL volumes every 6 hours. Six hundred microliters of medium was replaced by fresh cystine-deficient medium after 42 hours, 66 hours, and 84 hours of culture to keep the cumulative cystine concentration <120 μmol/L. After 84 hours, cells were pulsed with [3H]thymidine at 5 μCi/mL and harvested 18 hours later on glass fiber filters using an automatic cell harvester. [3H]Thymidine uptake was measured in a microplate scintillation counter (TopCount, Packard, Meriden, CT). Purified CD33+ LP-MO (5 × 105) and PB-MO, respectively, were collected in 300 μL lysis buffer of the MagNA Pure LC mRNA Isolation Kit I (Roche, Mannheim, Germany), and messenger RNA (mRNA) was isolated with the MagNA Pure LC instrument using the mRNA-I standard protocol (elution volume 50 μL); 8.2 μL mRNA was reverse transcribed using AMV-RT and oligo-p(dT)15 as primer (First Strand cDNA Synthesis Kit for RT-PCR; Roche) according to the manufacturer's instructions. The reaction mix was diluted to 500 μL and stored at −20°C until analysis. Primer sets specific for the sequences of 4F2hc and xCT optimized for the LightCycler (Roche) were provided by Search-LC (Heidelberg, Germany). The polymerase chain reaction (PCR) was performed with the LightCycler FastStart DNA Master Sybr Green I kit (Roche) according to the protocol provided. The number of complementary DNA (cDNA) transcripts was calculated from a standard curve as described previously, and the data were normalized according to the average expression of the housekeeping genes cyclophilin B and β-actin.19Autschbach F. Giese T. Gassler N. et al.Cytokine/chemokine messenger-RNA expression profiles in ulcerative colitis and Crohn's disease.Virchows Arch. 2002; 441: 500-513Google Scholar Values are presented as input-adjusted numbers of transcripts per microliter of cDNA. For RT-PCR analysis of gut tissue samples, 40-mg aliquots of transmural slices from frozen tissue blocks (−25°C) were homogenized in 400 μL lysis buffer with a Hybaid-ribolyser (AGS, Heidelberg, Germany). The mRNA was extracted using the MagNA Pure LC mRNA Isolation Kit II (Roche) and further processed as described above. An oligopeptide [KGQTQNFKDAFSGRDSSI] corresponding to amino acid residues 215–232 of the extracellular domain 3 of human xCT (SWISS-PROT accession number Q9UPY5 ) was conjugated to keyhole limpet hemocyanin via an additional C-terminus cysteine and was used for immunization of rabbits. The polyclonal anti-human xCT Ab was purified from rabbit serum by peptide affinity chromatography. All procedures were performed by Bethyl Laboratories (Montgomery, TX). Pelleted cells were lyzed in 50 μL cold lysis buffer containing 50 mmol/L Tris (pH 7.4), 165 mmol/L NaCl, 1% NP-40, 10 mmol/L EDTA, 10 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L PMSF, and a cocktail of protease inhibitors (Complete; Roche). Following 5 freeze-thaw cycles and addition of 2-ME-containing sample buffer, homogenates were heated (95°C, 5 minutes) and stored at −80°C. Protein samples were diluted to equal protein concentrations, separated by 12% SDS-PAGE, and transferred to a Hybond-P PVDF membrane (Amersham). Polyclonal rabbit anti-human xCT Ab, 1/1500, was used as the primary Ab. Specific binding was detected by a second-stage goat anti-rabbit HRP-conjugated Ab (Santa Cruz, Heidelberg, Germany), 1/2000, and visualized using the ECL Plus kit (Amersham). For analysis of xCT in unfractionated mucosa, fresh-frozen tissue samples were homogenized in TRI reagent (Sigma) using the Ultra Turrax equipment (IKA Labortechnik, Staufen, Germany). Immunoblotting was performed analogous to the procedure described above. The intracellular GSH level was determined using a mouse mAb directed against the adduct of GSH and N-ethylmaleimide (GS-NEM, IgG1; US Biological, Swampscott, MA) as previously described.20Palace G.P. Lawrence D.A. Phospholipid metabolism of lymphocytes with inhibited glutathione synthesis using L-buthionine-S,R-sulfoximine.Free Radic Biol Med. 1995; 18: 709-720Google Scholar This mAb does not detect oxidized glutathione or other thiol compounds. Cells were fixed in ice-cold 1% paraformaldehyde for 5 minutes, washed twice in cold PBS/0.5% bovine serum albumin containing 0.1% saponin, and resuspended in 10 mmol/L N-ethylmaleimide dissolved in methanol for 1 hour at room temperature. Cells were washed twice and then permeabilized using 0.1% saponin in PBS/10% human serum on ice for 30 minutes prior to incubation with the GS-NEM mAb, 1/1000, for 1 hour on ice. For negative controls, an isotype and concentration-matched mouse IgG1 mAb, 1/100 (Dianova), was used. A FITC-conjugated goat anti-mouse IgG1 Ab, 1/100 (Dianova), was used as secondary Ab. For double fluorescence staining, T cells were labelled with a PE-conjugated mouse anti-CD3 mAb (IgG1; BD) and analyzed using a FACSCalibur flow cytometer (BD). The following primary antibodies were used: mouse mAb against the adduct of GSH and N-ethylmaleimide, 1/40 and 1/20 for immunoenzyme and immunofluorescence staining, respectively (GS-NEM, clone 2Q2270, IgG1; US Biological); polyclonal rabbit anti-human xCT Ab, 1/100; mouse anti-human CD3 mAb, 1/5 (clone PS-1; IgG2a; Biogenex, San Ramon, CA); mouse anti-human CD11c mAb, 1/40 (clone KB90; IgG1; Dako, Hamburg, Germany); mouse anti-human CD14 mAb, 1/100 (clone MEM-18, IgG1; kindly provided by V. Horejsi, Prague, Czechoslovakia); mouse anti-human CD20 mAb, 1/50 (clone L-26, IgG2a; Dako); mouse anti-human CD68 mAb, 1/200 (clone KP-1, IgG1; Dako); mouse anti-human CD83 mAb, 1/100 (clone HB15e, IgG1; R&D Systems); mouse-anti-human CD138, 1/25 (clone MI15, IgG1; Dako). Isotype- and concentration-matched mouse control mAb as well as normal rabbit Ig (Dako, Dianova, R&D Systems) served as negative controls. Immunoenzyme staining of xCT was performed on 4-μm cryostat sections of fresh frozen tissues, which were postfixed in 2% paraformaldehyde and further processed by the paraformaldehyde-saponin-procedure in combination with the standard alkaline phosphatase antialkaline phosphatase technique (Dako) as previously published.19Autschbach F. Giese T. Gassler N. et al.Cytokine/chemokine messenger-RNA expression profiles in ulcerative colitis and Crohn's disease.Virchows Arch. 2002; 441: 500-513Google Scholar The primary Ab was added overnight at room temperature. A mouse anti-rabbit mAb, 1/50 (Dako), was used as a secondary reagent (30 minutes at room temperature). Naphthol AS-biphosphate (Sigma) with New-fuchsin (Merck, Darmstadt, Germany) served as the substrate for alkaline phosphatase. Immunoenzyme staining of GSH was performed on 2-μm paraffin sections of formalin-fixed mucosal biopsy specimens using the standard alkaline phosphatase antialkaline phosphatase procedure, except that the slides were first immersed in 10 mmol/L N-ethylmaleimide (Sigma) for 30 minutes at room temperature prior to addition of the mAb directed against the adduct of GSH and N-ethylmaleimide. GSH stainings were quantified as previously described.19Autschbach F. Giese T. Gassler N. et al.Cytokine/chemokine messenger-RNA expression profiles in ulcerative colitis and Crohn's disease.Virchows Arch. 2002; 441: 500-513Google Scholar Five hundred nucleated cells were counted in 2 different representative areas of the lamina propria (magnification, ×25) using a calibrated grid. Results are expressed as percent immunostained cells of the total number of nucleated cells. Double immunofluorescence staining was performed using pairwise combinations of primary mAb. Incubations were performed overnight at 4°C or at room temperature (GSH). For simultaneous detection of mouse mAb of different isotypes, a combination of biotinylated rabbit anti-mouse IgG1, 1/100 (Cytomed, Berlin, Germany), and sheep anti-mouse IgG2a, 1/300 (Binding Site, Schwetzingen, Germany), was used as secondary reagents (30 minutes at room temperature) followed by Cy-3-conjugated streptavidin, 1/1000 (red fluorescence; Dianova), and Cy2-conjugated donkey anti-sheep Ab, 1/50 (green fluorescence; Dianova), for 30 minutes. For simultaneous detection of rabbit and mouse primary reagents, a combination of biotinylated donkey anti-rabbit Ab, 1/200 (Cytomed, Dianova), and sheep anti-mouse Ab, 1/250 (Binding Site), served as secondary reagents. Single stainings were performed using Cy-3-conjugated streptavidin. Slides were viewed with a Laserscan microscope (Leica Microsystems, Mannheim, Germany). The Statistical Package for Social Sciences software (SPSS/PC, Chicago, IL) was used for data analysis. Results were expressed as means ± SD or as box plots. We performed statistical analysis between groups using the 2-tailed Mann-Whitney U test for unpaired samples, and P < .05 was considered statistically significant. We compared total intracellular glutathione concentrations in LP-T isolated from normal colon with those in autologous PB-T. Figure 1 demonstrates that the total glutathione content in LP-T is <50% of that measured in PB-T (P = .002). In contrast to autologous PB-MO, no secretion of cysteine was detectable in supernatants of LP-MO from normal gut with or without stimulation with lipopolysaccharide (Figure 2A). Production of cysteine requires cellular uptake of cystine via the cystine/glutamate exchange transporter and subsequent intracellular reduction of cystine to the monothiol cysteine. We investigated the transmembrane transport activity for cystine in lamina propria vs peripheral blood mononuclear cells. As shown in Figure 2B, the mean cellular uptake of [35S]cystine by LP-MO from normal gut was extremely low and indistinguishable from that by autologous T cells. In contrast, there was an avid uptake of [35S]cystine by autologous PB-MO (P < .01 vs LP-MO). Cystine uptake by PB-MO was Na+-independent and could be inhibited in the presence of a 50-fold excess of extracellular glutamate (2.5 mmol/L), thus verifying the specificity of the transport system xc− (cystine/glutamate exchange transporter). Note, however, that LP-MO possess a phagocytotic activity identical to that of autologous PB-MO (data not shown). The transport system xc− is a disulfide-linked heterodimer consisting of the functional subunit xCT and 4F2 heavy chain (4F2hc or CD98). 4F2hc, which is shared by several amino acid transporters, is required for membrane expression of the light chain xCT and induction of system xc− transport activity.21Bassi M.T. Gasol E. Manzoni M. et al.Identification and characterisation of human xCT that co-expresses with 4F2 heavy chain, the amino acid transport activity system xc−.Pflugers Arch. 2001; 442: 286-296Google Scholar The defective transmembrane transport activity for cystine in LP-MO is explained by the absence of xCT protein in contrast to PB-MO (Figure 2C). The differential xCT expression is apparently regulated at the mRNA level because gene expression of xCT in LP-MO was only ∼20% of that in PB-MO, whereas 4F2hc was normally expressed (Figure 2D). To exclude the possibility that the absence of xCT protein in resident LP-MO represents an artefact because of the isolation procedure, we performed in situ immunoenzyme stainings of xCT; Figure 3A shows that, in normal gut, immunoreactivity of lamina propria mononuclear cells for xCT is virtually negative, thus confirming the in vitro results. Considerable staining of epithelial cells, predominantly goblet cells, was caused by endogenous alkaline phosphatase activity because it was also observed in the negative control. In contrast, in IBD specimens, we detected an enhanced in situ expression of xCT by mononuclear cells within the inflamed lamina propria both in UC and CD (Figure 3A). Immunofluorescence stainings confirmed the enhanced xCT expression on lamina propria cells in IBD vs normal gut (Figure 3B). In these experiments, epithelia were either negative or showed a low-level expression at their apical membrane only. A major proportion of xCT+ lamina propria cells in IBD was found to coexpress CD68 in double immunofluorescence stainings and, thus, belong to the mononuclear phagocyte/macrophage lineage (Figure 4A). Similarly, CD14+ macrophages coexpressed xCT (Figure 4B). Positive xCT stainings also included a subset of CD83+ cells with dendritic morphology (Figure 4C). CD20+ B cells were found to express xCT within the inflamed lamina propria (Figure 4D), whereas CD138+ plasma cells see (Supplementary Figure 1A online at www.gastrojournal.org) and CD3+ LP-T were negative see (Supplementary Figure 1B online at www.gastrojournal.org). In isolated lymphoid follicles, CD11c+ dendritic cell populations showed immunoreactivity for xCT see (Supplementary Figure 1C online at www.gastrojournal.org). Because xCT expression was shown to be regulated at the transcriptional level in isolated cells (Figure 2D), we also quantitatively analyzed xCT mRNA in >100 extracts of transmural gut tissue samples. As shown in Figure 5A, tr
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