A Secreted Low--Molecular-Weight Protein From Helicobacter pylori Induces Cell-Cycle Arrest of T Cells
2005; Elsevier BV; Volume: 128; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2005.03.018
ISSN1528-0012
AutoresMarkus Gerhard, Christian Schmees, Petra Voland, N. Endres, Markus Sander, Wolfgang Reindl, Roland Rad, Madlene Oelsner, Thomas Decker, Martin Mempel, Ludger Hengst, Christian Prinz,
Tópico(s)Immune Cell Function and Interaction
ResumoBackground & aims: Although Helicobacter pylori is recognized by the human immune system, the bacteria are not eliminated and lead to a chronic inflammation of the gastric mucosa. Methods: We investigated the interaction of H pylori with human lymphocytes. T and B lymphocytes were isolated from H pylori–infected patients and stimulated with anti-CD3/CD28 or interleukin-6. Results: Proliferation of lymphocytes was abolished on co-incubation with different H pylori strains (1–5 bacteria/cell) or with protein extracts of culture supernatants. Inhibition of proliferation was independent of known virulence factors. The factor is a protein or protein complex with an apparent molecular weight between 30 and 60 kilodaltons, clearly distinct from VacA. Although antigen-specific activation of T cells (as shown by nuclear factor of activated T cells [NFAT]-activation, interferon-γ production, and CD25 or CD69 up-regulation) remained intact, cell-cycle analysis showed that S-phase entry of T cells was inhibited completely by H pylori. Consequently, stimulated T cells arrested in the G1 phase of the cell cycle. Western blot analysis showed markedly reduced phosphorylation of the retinoblastoma protein (pRb), suggesting inhibition of G1 cyclin-dependent kinase activity. In line with this, activities of cyclin D3 and cyclin E were down-regulated, and levels of the cyclin-dependent kinase inhibitor p27Kip1 were increased. Mouse embryonic fibroblasts deficient in p27 showed a decrease in H pylori–induced inhibition of cell proliferation, suggesting a central role for p27 in mediating H pylori–induced G1 arrest. Conclusions: Induction of cell-cycle arrest in lymphocytes may be of major significance for the chronic persistence of bacteria in the human stomach. Background & aims: Although Helicobacter pylori is recognized by the human immune system, the bacteria are not eliminated and lead to a chronic inflammation of the gastric mucosa. Methods: We investigated the interaction of H pylori with human lymphocytes. T and B lymphocytes were isolated from H pylori–infected patients and stimulated with anti-CD3/CD28 or interleukin-6. Results: Proliferation of lymphocytes was abolished on co-incubation with different H pylori strains (1–5 bacteria/cell) or with protein extracts of culture supernatants. Inhibition of proliferation was independent of known virulence factors. The factor is a protein or protein complex with an apparent molecular weight between 30 and 60 kilodaltons, clearly distinct from VacA. Although antigen-specific activation of T cells (as shown by nuclear factor of activated T cells [NFAT]-activation, interferon-γ production, and CD25 or CD69 up-regulation) remained intact, cell-cycle analysis showed that S-phase entry of T cells was inhibited completely by H pylori. Consequently, stimulated T cells arrested in the G1 phase of the cell cycle. Western blot analysis showed markedly reduced phosphorylation of the retinoblastoma protein (pRb), suggesting inhibition of G1 cyclin-dependent kinase activity. In line with this, activities of cyclin D3 and cyclin E were down-regulated, and levels of the cyclin-dependent kinase inhibitor p27Kip1 were increased. Mouse embryonic fibroblasts deficient in p27 showed a decrease in H pylori–induced inhibition of cell proliferation, suggesting a central role for p27 in mediating H pylori–induced G1 arrest. Conclusions: Induction of cell-cycle arrest in lymphocytes may be of major significance for the chronic persistence of bacteria in the human stomach. Helicobacter pylori is a gram-negative pathogen that selectively colonizes the human gastric mucosa and is prevalent in more than 50% of the world population. The infection mostly persists lifelong and has been implicated in the pathogenesis of gastric and duodenal ulcers, gastric mucosa-associated lymphoid-like tissue lymphoma, and gastric cancer.1Blaser M.J. Helicobacter pylori and gastric diseases.BMJ. 1998; 316: 1507-1510Crossref PubMed Scopus (247) Google Scholar A hallmark of H pylori infection is chronic active gastritis, characterized by dense infiltration of the mucosa by neutrophilic granulocytes, lymphocytes, and monocytes/macrophages. Several studies have provided evidence that T-helper type 1 cells are increased and activated during H pylori–associated gastritis, showing up-regulation of CD25 and CD69 in vivo.2Bamford K.B. Fan X. Crowe S.E. Leary J.F. Gourley W.K. Luthra G.K. Brooks E.G. Graham D.Y. Reyes V.E. Ernst P.B. Lymphocytes in the human gastric mucosa during Helicobacter pylori have a T helper cell 1 phenotype.Gastroenterology. 1998; 114: 482-492Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 3D'Elios M.M. Manghetti M. De Carli M. Costa F. Baldari C.T. Burroni D. Telford J.L. Romagnani S. Del Prete G. T helper 1 effector cells specific for Helicobacter pylori in the gastric antrum of patients with peptic ulcer disease.J Immunol. 1997; 158: 962-967PubMed Google Scholar, 4Stromberg E. Lundgren A. Edebo A. Lundin S. Svennerholm A.M. Lindholm C. Increased frequency of activated T-cells in the Helicobacter pylori-infected antrum and duodenum.FEMS Immunol Med Microbiol. 2003; 36: 159-168Crossref PubMed Scopus (47) Google Scholar, 5Quiding-Jarbrink M. Lundin B.S. Lonroth H. Svennerholm A.M. CD4+ and CD8+ T cell responses in Helicobacter pylori-infected individuals.Clin Exp Immunol. 2001; 123: 81-87Crossref PubMed Scopus (59) Google Scholar, 6Terres A.M. Pajares J.M. An increased number of follicles containing activated CD69+ helper T cells and proliferating CD71+ B cells are found in H. pylori-infected gastric mucosa.Am J Gastroenterol. 1998; 93: 579-583PubMed Google Scholar A strong humoral response to a variety of H pylori antigens also is elicited. Despite this inflammatory response, the infection is not cleared by the host immune system. Therefore, it appears that H pylori interferes with the immune system, but the distinct mechanisms remain obscure so far. Some studies have addressed this issue and described passive and active ways H pylori escape the immune response. Resistance of H pylori to phagocytosis has been reported and depends on virulence genes, such as virB7 and virB11, which encode components of the type IV secretion apparatus.7Allen L.A. Schlesinger L.S. Kang B. Virulent strains of Helicobacter pylori demonstrate delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages.J Exp Med. 2000; 191: 115-128Crossref PubMed Scopus (187) Google Scholar, 8Ramarao N. Gray-Owen S.D. Backert S. Meyer T.F. Helicobacter pylori inhibits phagocytosis by professional phagocytes involving type IV secretion components.Mol Microbiol. 2000; 37: 1389-1404Crossref PubMed Scopus (111) Google Scholar Zabaleta et al9Zabaleta J. McGee D.J. Zea A.H. Hernandez C.P. Rodriguez P.C. Sierra R.A. Correa P. Ochoa A.C. Helicobacter pylori arginase inhibits T cell proliferation and reduces the expression of the TCR zeta-chain (CD3zeta).J Immunol. 2004; 173: 586-593PubMed Google Scholar reported H pylori arginase to inhibit T-cell proliferation and reduce the expression of the T-cell receptor ζ chain. A proinflammatory peptide H2Bamford K.B. Fan X. Crowe S.E. Leary J.F. Gourley W.K. Luthra G.K. Brooks E.G. Graham D.Y. Reyes V.E. Ernst P.B. Lymphocytes in the human gastric mucosa during Helicobacter pylori have a T helper cell 1 phenotype.Gastroenterology. 1998; 114: 482-492Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar, 3D'Elios M.M. Manghetti M. De Carli M. Costa F. Baldari C.T. Burroni D. Telford J.L. Romagnani S. Del Prete G. T helper 1 effector cells specific for Helicobacter pylori in the gastric antrum of patients with peptic ulcer disease.J Immunol. 1997; 158: 962-967PubMed Google Scholar, 4Stromberg E. Lundgren A. Edebo A. Lundin S. Svennerholm A.M. Lindholm C. Increased frequency of activated T-cells in the Helicobacter pylori-infected antrum and duodenum.FEMS Immunol Med Microbiol. 2003; 36: 159-168Crossref PubMed Scopus (47) Google Scholar, 5Quiding-Jarbrink M. Lundin B.S. Lonroth H. Svennerholm A.M. CD4+ and CD8+ T cell responses in Helicobacter pylori-infected individuals.Clin Exp Immunol. 2001; 123: 81-87Crossref PubMed Scopus (59) Google Scholar, 6Terres A.M. Pajares J.M. An increased number of follicles containing activated CD69+ helper T cells and proliferating CD71+ B cells are found in H. pylori-infected gastric mucosa.Am J Gastroenterol. 1998; 93: 579-583PubMed Google Scholar, 7Allen L.A. Schlesinger L.S. Kang B. Virulent strains of Helicobacter pylori demonstrate delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages.J Exp Med. 2000; 191: 115-128Crossref PubMed Scopus (187) Google Scholar, 8Ramarao N. Gray-Owen S.D. Backert S. Meyer T.F. Helicobacter pylori inhibits phagocytosis by professional phagocytes involving type IV secretion components.Mol Microbiol. 2000; 37: 1389-1404Crossref PubMed Scopus (111) Google Scholar, 9Zabaleta J. McGee D.J. Zea A.H. Hernandez C.P. Rodriguez P.C. Sierra R.A. Correa P. Ochoa A.C. Helicobacter pylori arginase inhibits T cell proliferation and reduces the expression of the TCR zeta-chain (CD3zeta).J Immunol. 2004; 173: 586-593PubMed Google Scholar, 10Putsep K. Branden C.I. Boman H.G. Normark S. Antibacterial peptide from H. pylori.Nature. 1999; 398: 671-672Crossref PubMed Scopus (178) Google Scholar, 11Betten A. Bylund J. Cristophe T. Boulay F. Romero A. Hellstrand K. Dahlgren C. A proinflammatory peptide from Helicobacter pylori activates monocytes to induce lymphocyte dysfunction and apoptosis.J Clin Invest. 2001; 108: 1221-1228Crossref PubMed Scopus (117) Google Scholar, 12Akhiani A.A. Pappo J. Kabok Z. Schon K. Gao W. Franzen L.E. Lycke N. Protection against Helicobacter pylori infection following immunization is IL-12-dependent and mediated by Th1 cells.J Immunol. 2002; 169: 6977-6984PubMed Google Scholar, 13Boncristiano M. Paccani S.R. Barone S. Ulivieri C. Patrussi L. Ilver D. Amedei A. D'Elios M.M. Telford J.L. Baldari C.T. The Helicobacter pylori vacuolating toxin inhibits T cell activation by two independent mechanisms.J Exp Med. 2003; 198: 1887-1897Crossref PubMed Scopus (243) Google Scholar, 14Gebert B. Fischer W. Weiss E. Hoffmann R. Haas R. Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation.Science. 2003; 301: 1099-1102Crossref PubMed Scopus (461) Google Scholar, 15Sundrud M.S. Torres V.J. Unutmaz D. Cover T.L. Inhibition of primary human T cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion.Proc Natl Acad Sci U S A. 2004; 101: 7727-7732Crossref PubMed Scopus (213) Google Scholar, 16Wang J. Brooks E.G. Bamford K.B. Denning T.L. Pappo J. Ernst P.B. Negative selection of T cells by Helicobacter pylori as a model for bacterial strain selection by immune evasion.J Immunol. 2001; 167: 926-934PubMed Google Scholar, 17Karttunen R. Blood lymphocyte proliferation, cytokine secretion and appearance of T cells with activation surface markers in cultures with Helicobacter pylori Comparison of the responses of subjects with and without antibodies to H. pylori.Clin Exp Immunol. 1991; 83: 396-400Crossref PubMed Scopus (87) Google Scholar, 18Fan X.G. Yakoob J. Fan X.J. Keeling P.W. Effect of IL-4 on peripheral blood lymphocyte proliferation implication in immunopathogenesis of H.pylori infection. Immunol Lett. 1995; 48: 45-48Crossref PubMed Scopus (9) Google Scholar, 19Knipp U. Birkholz S. Kaup W. Opferkuch W. Immune suppressive effects of Helicobacter pylori on human peripheral blood mononuclear cells.Med Microbiol Immunol (Berl). 1993; 182: 63-76Crossref PubMed Scopus (36) Google Scholar, 20Knipp U. Birkholz S. Kaup W. Mahnke K. Opferkuch W. Suppression of human mononuclear cell response by Helicobacter pylori effects on isolated monocytes and lymphocytes.FEMS Immunol Med Microbiol. 1994; 8: 157-166Crossref PubMed Scopus (36) Google Scholar of H pylori10Putsep K. Branden C.I. Boman H.G. Normark S. Antibacterial peptide from H. pylori.Nature. 1999; 398: 671-672Crossref PubMed Scopus (178) Google Scholar has been shown to induce lymphocytic dysfunction by activating monocytes to produce reactive oxygen radicals.11Betten A. Bylund J. Cristophe T. Boulay F. Romero A. Hellstrand K. Dahlgren C. A proinflammatory peptide from Helicobacter pylori activates monocytes to induce lymphocyte dysfunction and apoptosis.J Clin Invest. 2001; 108: 1221-1228Crossref PubMed Scopus (117) Google Scholar These data emphasize the interaction of the bacteria with the nonspecific immune response; however, a specific T-cell response appears to be decisive for elimination of the bacteria because vaccination trials have failed in mice deficient of T cells or interferon-γ (IFN-γ).12Akhiani A.A. Pappo J. Kabok Z. Schon K. Gao W. Franzen L.E. Lycke N. Protection against Helicobacter pylori infection following immunization is IL-12-dependent and mediated by Th1 cells.J Immunol. 2002; 169: 6977-6984PubMed Google Scholar In this context, 2 recent in vitro studies suggested that the presence of high concentrations of VacA (250 μg/mL whole bacterial protein or ≤5 μg/mL VacA) interferes with the activation of nuclear factor of activated T cells (NFAT) in T lymphocytes, leading to impaired interleukin-2 (IL-2) secretion.13Boncristiano M. Paccani S.R. Barone S. Ulivieri C. Patrussi L. Ilver D. Amedei A. D'Elios M.M. Telford J.L. Baldari C.T. The Helicobacter pylori vacuolating toxin inhibits T cell activation by two independent mechanisms.J Exp Med. 2003; 198: 1887-1897Crossref PubMed Scopus (243) Google Scholar, 14Gebert B. Fischer W. Weiss E. Hoffmann R. Haas R. Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation.Science. 2003; 301: 1099-1102Crossref PubMed Scopus (461) Google Scholar However, this observation could not be confirmed by Sundrud et al,15Sundrud M.S. Torres V.J. Unutmaz D. Cover T.L. Inhibition of primary human T cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion.Proc Natl Acad Sci U S A. 2004; 101: 7727-7732Crossref PubMed Scopus (213) Google Scholar who found that VacA at 10 μg/mL inhibited proliferation of T lymphocytes without interfering with NFAT activation. Further, several studies have shown that H pylori, mostly at high bacteria per cell ratios (multiplicity of infection [MOI], 100–300), induced apoptosis of T cells,16Wang J. Brooks E.G. Bamford K.B. Denning T.L. Pappo J. Ernst P.B. Negative selection of T cells by Helicobacter pylori as a model for bacterial strain selection by immune evasion.J Immunol. 2001; 167: 926-934PubMed Google Scholar impaired the proliferation of lymphocytes,17Karttunen R. Blood lymphocyte proliferation, cytokine secretion and appearance of T cells with activation surface markers in cultures with Helicobacter pylori Comparison of the responses of subjects with and without antibodies to H. pylori.Clin Exp Immunol. 1991; 83: 396-400Crossref PubMed Scopus (87) Google Scholar, 18Fan X.G. Yakoob J. Fan X.J. Keeling P.W. Effect of IL-4 on peripheral blood lymphocyte proliferation implication in immunopathogenesis of H.pylori infection. Immunol Lett. 1995; 48: 45-48Crossref PubMed Scopus (9) Google Scholar, 19Knipp U. Birkholz S. Kaup W. Opferkuch W. Immune suppressive effects of Helicobacter pylori on human peripheral blood mononuclear cells.Med Microbiol Immunol (Berl). 1993; 182: 63-76Crossref PubMed Scopus (36) Google Scholar, 20Knipp U. Birkholz S. Kaup W. Mahnke K. Opferkuch W. Suppression of human mononuclear cell response by Helicobacter pylori effects on isolated monocytes and lymphocytes.FEMS Immunol Med Microbiol. 1994; 8: 157-166Crossref PubMed Scopus (36) Google Scholar or inhibited the proliferation of murine splenocytes.21Chen W. Shu D. Chadwick V.S. Inhibition of mitogen-induced murine lymphocyte proliferation by Helicobacter pylori cell-free extract.J Gastroenterol Hepatol. 2000; 15: 1000-1006Crossref PubMed Scopus (9) Google Scholar These studies suggested a direct interaction of the bacteria with T cells; however, the underlying mechanism of an inhibition of lymphocyte proliferation has not been elucidated, and the role of bacterial virulence factors for such effects has not been clarified. Therefore, we decided to investigate the effect of various H pylori strains on the proliferation of human lymphocytes. Here, we show evidence for a direct inhibition of lymphocyte proliferation by a soluble and possibly secreted H pylori protein. The protein prevents lymphocyte proliferation at very low and thus probably physiologic concentrations. The factor arrests human lymphocytes in the G1 phase of the cell cycle, thus providing a potentially crucial mechanism for immune evasion. RPMI 1640, Dulbecco's modified Eagle medium, fetal calf serum (FCS), and Hank's balanced salt solution were purchased from Gibco (Karlsruhe, Germany). [3H]-thymidine was obtained from Amersham Biosciences (Freiburg, Germany). Cell proliferation was stimulated with phytohemagglutinin (PHA) at 5–10 μg/mL, phorbol 12-myristate-13-acetate at 100 nmol/L, OKT3 at 1 μg/mL, or Ca-Ionophore at 10−5 mol/L (all purchased from Sigma, Munich, Germany). Monoclonal antibodies for subtyping of surface markers (CD3, CD4, CD8, CD14, and CD19) were obtained from Diatec (Oslo, Norway), and anti-CD16 antibody from Merck Biosciences. Anti–CD8–fluorescein isothiocyanate, anti–CD69-phycorythrin (PE), and secondary antibodies were obtained from Becton-Dickinson. Antibodies against IL-1β, tumor necrosis factor-α, and transforming growth factor-β used for inhibition experiments were purchased from Dianova (Hamburg, Germany). LT-12 cells were obtained through Annick Lim (Institut Pasteur, Paris with kind permission of E. Dufour, Institut Gustav-Roissy, Villejuif, France). The MART-127–35 peptide AAGIGILTV was obtained from Biosyntan (Berlin, Germany). NG-monomethyl-L-arginine (L-NMMA) was obtained from Calbiochem (Merck, Darmstadt, Germany). H pylori strains and mutants used in this study have been described earlier.22Gerhard M. Lehn N. Neumayer N. Boren T. Rad R. Schepp W. Miehlke S. Classen M. Prinz C. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin.Proc Natl Acad Sci U S A. 1999; 96: 12778-12783Crossref PubMed Scopus (530) Google Scholar Strains M26 and L76 are clinical isolates lacking the cag pathogenicity island. Strains G27 and G27▵cagE were obtained from A. Covacci (IRIS, Siena, Italy), strain P12 was obtained from S. Odenbreit (Munich, Germany); strains TX30a, 26695, and J99 were obtained from N. Lehn (University of Regensburg, Regensburg, Germany). Strain 60190▵VacA was kindly provided by R. Atherton (University of Nottingham, United Kingdom). Knock-out mutants for BabA, SabA, OipA, and CagA were kindly provided by J. Kusters (Erasmus MC, Rotterdam, The Netherlands). Strains were cultured on Wilkins-Chalgren or brain-heart infusion agar plates supplemented with Dent supplement antibiotic mix (Oxoid, Wesel, Germany) as previously described.22Gerhard M. Lehn N. Neumayer N. Boren T. Rad R. Schepp W. Miehlke S. Classen M. Prinz C. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin.Proc Natl Acad Sci U S A. 1999; 96: 12778-12783Crossref PubMed Scopus (530) Google Scholar Liquid culture of H pylori was performed in brain-heart infusion broth supplemented with 10% FCS. Escherichia coli were cultured on Luria broth agar plates containing 5% sheep blood (Becton-Dickinson), C jejuni were cultured under microaerophil conditions on Campylsel agar plates (BioMerieux, Nürtingen, Germany). Strains were genotyped using previously published primers for vacA, cagA, and babA.22Gerhard M. Lehn N. Neumayer N. Boren T. Rad R. Schepp W. Miehlke S. Classen M. Prinz C. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin.Proc Natl Acad Sci U S A. 1999; 96: 12778-12783Crossref PubMed Scopus (530) Google Scholar VacA mutants were analyzed by polymerase chain reaction using the primers: vacA797fw: 5′-ATGGAAATACAACAAACACA-3′; vacA1397rev:5′-CTCCAGAACCCACACGATT-3′. H pylori strains were grown on plates for 48 hours, washed 3 times in phosphate-buffered saline (PBS), and adjusted to OD600nm = 1 (corresponding to ∼2 × 108 bacteria/mL). To disrupt the H pylori cells, bacteria were pelleted by centrifugation, resuspended in double distilled H2O (ddH2O), and sonicated with a Branson sonifier (Heusenstamm, Germany) at 20-second intervals (50% pulses) for 3 minutes on ice. The lysate was centrifuged in a microfuge at 15,000 rpm for 10 minutes at 4°C to remove remaining cells and debris. To produce H pylori water extracts (HPWE), bacteria were incubated in PBS for 2 hours and pelleted by subsequent centrifugation steps at 3000×g and 10,000×g to remove bacteria and membranes, respectively. Protein content of the supernatants was measured by Bradford assay (Bio-Rad Laboratories, Richmond, VA) with bovine serum albumin as standard and stored at −80°C. HPWE were prepared as described earlier and concentrated using ultrafiltration (Amicon Ultra; Millipore, Bedford, MA). Samples containing 500 μg of protein were loaded on a Superdex 200 10/300 column (Amersham Pharmacia Biotech) and eluted with degassed PBS at 4°C. For molecular-weight estimation of eluted peaks, standard proteins β-amylase (200 kilodaltons), alcohol dehydrogenase (150 kilodaltons), bovine serum albumin (66 kilodaltons), and carbonic anhydrase (29 kilodaltons) were used. During each run 50 fractions (.5 mL each) were collected and subsequently screened for inhibition of cell proliferation by [3H]-thymidine–incorporation assay (using 1/50 = 10 μL of each fraction). Peripheral blood lymphocytes (PBLs) were isolated from buffy coats or heparinized peripheral venous blood from (H pylori–infected or–uninfected) healthy volunteers by density-gradient centrifugation with Ficoll-Paque Plus (Amersham Bioscience) for 40 minutes at 400×g. The interphases were washed twice with Ca2+-and Mg2+-free PBS and resuspended in RPMI 1640/10% FCS containing less than 100 pg/mL endotoxin as determined by LAL-assay (LAL-QCL-1000, BioWhittaker Europe, Verviers, Belgium). T cells were isolated by positive selection using anti-CD3–coated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) or negative selection of non–T cells (Pan T-cell Isolation Kit; Miltenyi Biotech). Similarly, CD4+ and CD8+ cells were purified by positive selection using anti-CD4− and anti-CD8-coated magnetic beads. B cells were isolated by positive selection using anti-CD19–coated magnetic beads after removal of T cells. Jurkat T cells, cultured in RPMI 1640 with 10% FCS, were transfected with a NFAT–green fluorescent protein expression vector (kindly provided by R. Kehlenbach, Heidelberg, Germany).23Kehlenbach R.H. Dickmanns A. Gerace L. Nucleocytoplasmic shuttling factors including Ran and CRM1 mediate nuclear export of NFAT In vitro.J Cell Biol. 1998; 141: 863-874Crossref PubMed Scopus (143) Google Scholar Stable clones were selected with G418. For stimulation, 5 × 105 cells were seeded in 6-well plates and stimulated with phorbol 12-myristate-13-acetate and Ca-Ionophore alone or in parallel with HPWE at indicated concentrations. Localization of NFAT-green fluorescent protein fusion protein was visualized by confocal laser scanner microscopy on a Zeiss LSM5 microscope (Carl Zeiss, Jena, Germany). To determine NFAT activation, the SIINFEKL/Kb-specific B3Z/NFAT-lacZ T-cell hybridoma subline was used,24Shastri N. Gonzalez F. Endogenous generation and presentation of the ovalbumin peptide/Kb complex to T cells.J Immunol. 1993; 150: 2724-2736PubMed Google Scholar in which activation of NFAT drives β-galactosidase transcription. Cells were stimulated for 24 hours as described,25Maurer T. Heit A. Hochrein H. Ampenberger F. O'Keeffe M. Bauer S. Lipford G.B. Vabulas R.M. Wagner H. CpG-DNA aided cross-presentation of soluble antigens by dendritic cells.Eur J Immunol. 2002; 32: 2356-2364Crossref PubMed Scopus (155) Google Scholar lysed by addition of 100 μL Z-buffer (100 mmol/L 2-mercaptoethanol, 9 mmol/L MgCl2, .125% Nonidet P-40, .15 mmol/L chlorophenol red β-galactoside [Merck] in PBS), and absorption of individual wells was read using a 96-well plate reader (Dynex, Sussex, UK) at 570 nm, with 650 nm as the reference wave length. p27+/+ and p27−/− mouse embryonic fibroblasts (MEFs) were maintained in Dulbecco's modified Eagle medium supplemented with 4.5 g/L glucose, 1 mmol/L sodium pyruvate (Sigma, Munich, Germany), and 10% FCS. For proliferation assays MEFs were synchronized by serum starvation, plated at 8 × 102 cells/well in 96-well plates, and incubated with or without HPWE in the presence of serum for 48 hours. 105 PBL, MEFs, or purified T cells (>98% CD3+) were cultured in 96-well flat-bottom plates in complete RPMI 1640/10% FCS. Cells were stimulated in triplicate with PHA (5–10 μg/mL) and 20 U/mL of recombinant human IL-2 (Sigma, Munich, Germany) or anti-CD3/CD28 beads (Dynal Biotech, Hamburg, Germany), respectively, for 48–72 hours. Different H pylori strains or HPWE were added at the time of stimulation at indicated concentrations. In all experiments except as shown in Figure 1, HPWEs were used for inhibition experiments. Clonal expansion of T cells was determined after 48 hours by [3H]-thymidine incorporation measured by liquid scintillation counting in a Packard Direkt Beta Counter Matrix 9600 (Packard Instrument Co, Downer's Grove, IL). The T-cell clone LT-12 specifically recognizes MART-127–35 and is restricted by HLA.A2.26Dufour E. Carcelain G. Gaudin C. Flament C. Avril M.F. Faure F. Diversity of the cytotoxic melanoma-specific immune response some CTL clones recognize autologous fresh tumor cells and not tumor cell lines.J Immunol. 1997; 158: 3787-3795PubMed Google Scholar To screen for LT-12 activation, 105 HLA-A2 monocyte-derived dendritic cells that had been obtained by IL-4/granulocyte-macrophage colony–stimulating factor–stimulation following standard protocols were loaded with 10 μg/mL MART-127–35 peptide. After treatment with Mitomycin C (50 μg/mL) for 2 hours, the cells were washed and incubated with 2 × 105 LT-12 cells in 200 μL RPMI 1640 medium for 60 hours in 96-well plates. Afterward, the supernatant was aspirated and stored at −20°C for IFN-γ analysis. The cells were stained with anti-CD8/anti-CD69 to evaluate the percentage of activated CD8+ cells. IFN-γ and IL-2 in the supernatant were determined using a commercially available enzyme-linked immunosorbent assay from Biocarta (Hamburg, Germany). Cell-cycle analysis was performed by propidium iodide staining using the CycleTest Plus DNA Reagent Kit (Becton-Dickinson), and subsequent fluorescence-activated cell sorter (FACS) analysis, using a Becton-Dickinson FACScan flow cytometer, acquiring 5000 events. Data were analyzed using the Cell Quest software package (Becton-Dickinson). To identify fragmented DNA, a terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay (Roche, Mannheim, Germany) was performed and assessed by flow cytometry. Annexin V staining was performed using a PE-coupled antibody (Becton-Dickinson). T cells were collected 24, 48, and 72 hours after stimulation and resuspended in standard lysis buffer for 20 minutes at 4°C. Lysates were centrifuged at 12,000 rpm for 20 minutes and supernatants were collected. Total extracts (30 μg/lane) were subjected to 12% glycin-sodium dodecyl sulfate–polyacrylamide gel electrophoresis (7.5% for retinoblastoma [Rb]) and electrotransfer was performed onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Eschborn, Germany). Detection was performed as described previously.27Gerhard M. Neumayer N. Presecan-Siedel E. Zanner R. Lengyel E. Cramer T. Hocker M. Prinz C. Gastrin induces expression and promoter activity of the vesicular monoamine transporter subtype 2.Endocrinology. 2001; 142: 3663-3672Crossref PubMed Scopus (30) Google Scholar Cell-cycle regulating proteins were detected by monoclonal antibodies specific for p27, cyclin D2, cyclin D3, and Rb (BD Pharmingen, Heidelberg, Germany), cyclin-dependent kinase (cdk)2, cdk4, cyclin A, and cyclin E (Santa Cruz Biotechnology, Santa Cruz, CA), and actin (Sigma, Deisenhofen, Germany). All primary antibodies were used at dilutions of 1:2000. Blots were developed using Super Signal chemiluminescent substrates from Perbio Sciences (Dortmund, Germany). Data are expressed as mean ± SD if not indicated otherwise. For statistical evaluation, the Student t test was used. We first determined the interaction of H pylori with lymphocytes at low bacterial concentrations (MOI < 10), investigating the influence of H pylori on proliferation of PBL after stimulation with PHA. PHA induces nonselective activation of T cells. As shown in Figure 1A, PHA induced a strong proliferation in PBL (basal), and addition of H pylori at MOI of 1–10 completely abolished the proliferation. This effect also was observed if the bacteria were added 24 hours after initial stimulation, and was independent of the type of stimulant used (PHA, phorbol 12-myristate-13-acetate, or OKT3, not shown). In contrast, E coli or Campylobacter jejuni (not shown) were ineffective at the same concentrations. The inhibition of proliferation was observed by addition of various strain types and was independent of the presence of the cag pathogenicity island or the virulence factor VacA because the Tx30a strain (deficient in the cag pathogenicity island) and other type II strains (not shown) were equally effective (Figure 1B), indicating that the putative factor is present in most, if not all, H pylori strains. The inhibitory activity was retained when bacteria were killed by the addition of antibiotics (not shown) or sonication. After centrifugation, the inhibitory effec
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