Manifestation of Spontaneous and Early Autoimmune Gastritis in CCR7-Deficient Mice
2011; Elsevier BV; Volume: 179; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.04.012
ISSN1525-2191
AutoresSusann Winter, Armin Rehm, Katharina Wichner, Tobias Scheel, Arvind Batra, Britta Siegmund, Claudia Berek, Martin Lipp, Uta E. Höpken,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoAutoimmune gastritis is a common autoimmune disorder characterized by chronic inflammatory cell infiltrates, atrophy of the corpus and fundus, and the occurrence of autoantibodies to parietal cell antigen. In CCR7-deficient mice, autoimmune gastritis developed spontaneously and was accompanied by metaplasia of the gastric mucosa and by the formation of tertiary lymphoid organs at gastric mucosal sites. T cells of CCR7-deficient mice showed an activated phenotype in the gastric mucosa, mesenteric lymph nodes, and peripheral blood. In addition, elevated serum IgG levels specific to gastric parietal cell antigen were detected. Because the role of organized lymphocytic aggregates at this inflammatory site is not completely understood, we first analyzed the cellular requirements for the formation of these structures. Autoreactive CD4+ T cells were pivotal for tertiary lymphoid follicle formation, most likely in cooperation with dendritic cells, macrophages, and B cells. Second, we analyzed the necessity of secondary lymph nodes and tertiary lymphoid organs for the development of autoimmune gastritis using CCR7 single- and CCR7/lymphotoxin α double-deficient mice. Strikingly, manifestation of autoimmune gastritis was observed in the absence of secondary lymph nodes and preceded the development of tertiary lymphoid organs. Taken together, these findings identify an inflammatory process where gastric autoreactive T cells independent of organized tertiary lymphoid organs and classic lymph nodes can induce and maintain autoimmune gastritis. Autoimmune gastritis is a common autoimmune disorder characterized by chronic inflammatory cell infiltrates, atrophy of the corpus and fundus, and the occurrence of autoantibodies to parietal cell antigen. In CCR7-deficient mice, autoimmune gastritis developed spontaneously and was accompanied by metaplasia of the gastric mucosa and by the formation of tertiary lymphoid organs at gastric mucosal sites. T cells of CCR7-deficient mice showed an activated phenotype in the gastric mucosa, mesenteric lymph nodes, and peripheral blood. In addition, elevated serum IgG levels specific to gastric parietal cell antigen were detected. Because the role of organized lymphocytic aggregates at this inflammatory site is not completely understood, we first analyzed the cellular requirements for the formation of these structures. Autoreactive CD4+ T cells were pivotal for tertiary lymphoid follicle formation, most likely in cooperation with dendritic cells, macrophages, and B cells. Second, we analyzed the necessity of secondary lymph nodes and tertiary lymphoid organs for the development of autoimmune gastritis using CCR7 single- and CCR7/lymphotoxin α double-deficient mice. Strikingly, manifestation of autoimmune gastritis was observed in the absence of secondary lymph nodes and preceded the development of tertiary lymphoid organs. Taken together, these findings identify an inflammatory process where gastric autoreactive T cells independent of organized tertiary lymphoid organs and classic lymph nodes can induce and maintain autoimmune gastritis. Autoimmunity is often accompanied by the development of tertiary lymphoid organs (TLOs) in the target organ. TLO formation has been reported in inflamed tissues of various autoimmune diseases, including rheumatoid arthritis, Sjögren's syndrome, type 1 diabetes mellitus, and autoimmune gastritis (AIG).1Kim H.J. Berek C. B cells in rheumatoid arthritis.Arthritis Res. 2000; 2: 126-131Crossref PubMed Scopus (100) Google Scholar, 2Barone F. Bombardieri M. Rosado M.M. Morgan P.R. Challacombe S.J. De Vita S. Carsetti R. Spencer J. Valesini G. Pitzalis C. CXCL13. CCL21, and CXCL12 expression in salivary glands of patients with Sjögren's syndrome and MALT lymphoma: association with reactive and malignant areas of lymphoid organization.J Immunol. 2008; 180: 5130-5140PubMed Google Scholar, 3Wu Q. Salomon B. Chen M. Wang Y. Hoffman L.M. Bluestone J.A. Fu Y.X. Reversal of spontaneous autoimmune insulitis in nonobese diabetic mice by soluble lymphotoxin receptor.J Exp Med. 2001; 193: 1327-1332Crossref PubMed Scopus (104) Google Scholar, 4Kendall P.L. Yu G. Woodward E.J. Thomas J.W. Tertiary lymphoid structures in the pancreas promote selection of B lymphocytes in autoimmune diabetes.J Immunol. 2007; 178: 5643-5651Crossref PubMed Scopus (79) Google Scholar, 5Katakai T. Hara T. Sugai M. Gonda H. Shimizu A. Th1-biased tertiary lymphoid tissue supported by CXC chemokine ligand 13-producing stromal network in chronic lesions of autoimmune gastritis.J Immunol. 2003; 171: 4359-4368PubMed Google Scholar Although TLOs share numerous functional characteristics with secondary lymphoid organs (SLOs), their unique function in autoimmune disease pathogenesis is still unclear.6Drayton D.L. Liao S. Mounzer R.H. Ruddle N.H. Lymphoid organ development: from ontogeny to neogenesis.Nat Immunol. 2006; 7: 344-353Crossref PubMed Scopus (541) Google Scholar, 7Carragher D.M. Rangel-Moreno J. Randall T.D. Ectopic lymphoid tissues and local immunity.Semin Immunol. 2008; 20: 26-42Crossref PubMed Scopus (216) Google Scholar More specifically, it remains to be elucidated whether these structures are relevant for priming of T cells, development of autoantibody-producing plasma cells, and accumulation of autoantigen-specific T- and B-cell clones. In a murine AIG model induced by neonatal thymectomy, Katakai and colleagues found ectopic lymphoid organization in the chronic gastric lesions. Number and size of ectopic gastric lymphoid clusters could be correlated with serum autoantibody titers, although development of typical germinal centers (GCs) was rare in this AIG mouse model.5Katakai T. Hara T. Sugai M. Gonda H. Shimizu A. Th1-biased tertiary lymphoid tissue supported by CXC chemokine ligand 13-producing stromal network in chronic lesions of autoimmune gastritis.J Immunol. 2003; 171: 4359-4368PubMed Google Scholar, 8Katakai T. Shimizu A. Undesired meeting of lymphocytes: organ-specific infiltration and the organization of ectopic lymphoid tissue in a murine experimental autoimmune gastritis.Immunol Lett. 2008; 118: 103-109Crossref PubMed Scopus (2) Google Scholar In a nonobese diabetic mouse model of type 1 diabetes, the presence of GCs, a skewed repertoire, and somatic hypermutation of B-cell receptors was described in islet TLOs, which implies that T- and B-cell interactions take place at the inflamed site.4Kendall P.L. Yu G. Woodward E.J. Thomas J.W. Tertiary lymphoid structures in the pancreas promote selection of B lymphocytes in autoimmune diabetes.J Immunol. 2007; 178: 5643-5651Crossref PubMed Scopus (79) Google Scholar More recently, a CXCL13-dependent B lymphocyte lodging within islet TLOs was shown. On CXCL13 blockade, B-lymphocyte organization in TLOs was completely abrogated.9Henry RA, Kendall PL: CXCL13 blockade disrupts B lymphocyte organization in tertiary lymphoid structures without altering B cell receptor bias or preventing diabetes in nonobese diabetic mice. J Immunol 185:1460–1465Google Scholar However, induction of a skewed B-cell repertoire, occurrence of somatic hypermutation, and finally manifestation of diabetes were maintained. Thus, the authors concluded that the significance of organized TLOs in modulating autoimmune responses remains questionable. In contrast, other studies suggested that formation of TLOs in affected organs contributes essentially to the pathogenesis of human autoimmune diseases by enhancing the efficiency of autoantigen presentation at the site of inflammation. In this process, autoreactive T lymphocytes and autoantibody-producing B cells could be generated.7Carragher D.M. Rangel-Moreno J. Randall T.D. Ectopic lymphoid tissues and local immunity.Semin Immunol. 2008; 20: 26-42Crossref PubMed Scopus (216) Google Scholar Thus, TLOs may play a role in maintaining an autoimmune response against persistent self-antigens and selection of autoreactive B and T cells.4Kendall P.L. Yu G. Woodward E.J. Thomas J.W. Tertiary lymphoid structures in the pancreas promote selection of B lymphocytes in autoimmune diabetes.J Immunol. 2007; 178: 5643-5651Crossref PubMed Scopus (79) Google Scholar However, the contribution of autoantibodies to disease progression is still controversially discussed. There is evidence that lymphoid neogenesis is associated with local antibody production but not with autoimmune activity or severity.7Carragher D.M. Rangel-Moreno J. Randall T.D. Ectopic lymphoid tissues and local immunity.Semin Immunol. 2008; 20: 26-42Crossref PubMed Scopus (216) Google Scholar AIG represents an organ-specific autoimmune disease in which lymphocytes massively infiltrate the gastric mucosal (GM) tissue, which eventually leads to gastric lymphoid neogenesis. Loss of gastric parietal and zymogenic cells and the prevalence of circulating autoantibodies to gastric parietal antigen are specific characteristics.10van Driel I.R. Baxter A.G. Laurie K.L. Zwar T.D. La Gruta N.L. Judd L.M. Scarff K.L. Silveira P.A. Gleeson P.A. Immunopathogenesis, loss of T cell tolerance and genetics of autoimmune gastritis.Autoimmun Rev. 2002; 1: 290-297Crossref PubMed Scopus (37) Google Scholar The morphology of mouse gastritis shares many pathohistologic and immunologic characteristics with the human disease and can be induced in a variety of experimental approaches, including lymphopenic and nonlymphopenic AIG models, or in T-cell receptor α-subunit and T-cell receptor β-subunit transgenic mice.11Field J. Biondo M.A. Murphy K. Alderuccio F. Toh B.H. Experimental autoimmune gastritis: mouse models of human organ-specific autoimmune disease.Int Rev Immunol. 2005; 24: 93-110Crossref PubMed Scopus (20) Google Scholar Models of spontaneous AIG disease pathogenesis are rare, and disease incidence is often very low.11Field J. Biondo M.A. Murphy K. Alderuccio F. Toh B.H. Experimental autoimmune gastritis: mouse models of human organ-specific autoimmune disease.Int Rev Immunol. 2005; 24: 93-110Crossref PubMed Scopus (20) Google Scholar, 12Alderuccio F. Toh B.H. Spontaneous autoimmune gastritis in C3H/He mice: a new mouse model for gastric autoimmunity.Am J Pathol. 1998; 153: 1311-1318Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar We report the development of an early, spontaneous AIG with almost complete penetrance in mice lacking the homeostatic chemokine receptor CCR7. CCR7 is a major lymph node–homing receptor for lymphocytes and dendritic cells (DCs), and mice that lack CCR7 display not only a delayed induction of adaptive immune responses13Forster R. Schubel A. Breitfeld D. Kremmer E. Renner-Muller I. Wolf E. Lipp M. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.Cell. 1999; 99: 23-33Abstract Full Text Full Text PDF PubMed Scopus (1917) Google Scholar, 14Hopken U.E. Droese J. Li J.P. Joergensen J. Breitfeld D. Zerwes H.G. Lipp M. The chemokine receptor CCR7 controls lymph node-dependent cytotoxic T cell priming in alloimmune responses.Eur J Immunol. 2004; 34: 461-470Crossref PubMed Scopus (46) Google Scholar but also impaired lymphocyte recirculation through nonlymphoid organs.15Debes G.F. Arnold C.N. Young A.J. Krautwald S. Lipp M. Hay J.B. Butcher E.C. Chemokine receptor CCR7 required for T lymphocyte exit from peripheral tissues.Nat Immunol. 2005; 6: 889-894Crossref PubMed Scopus (385) Google Scholar, 16Hopken U.E. Wengner A.M. Loddenkemper C. Stein H. Heimesaat M.M. Rehm A. Lipp M. CCR7 deficiency causes ectopic lymphoid neogenesis and disturbed mucosal tissue integrity.Blood. 2007; 109: 886-895Crossref PubMed Scopus (55) Google Scholar Lack of CCR7 is associated with a moderate multiorgan autoimmunity17Davalos-Misslitz A.C. Rieckenberg J. Willenzon S. Worbs T. Kremmer E. Bernhardt G. Forster R. Generalized multi-organ autoimmunity in CCR7-deficient mice.Eur J Immunol. 2007; 37: 613-622Crossref PubMed Scopus (97) Google Scholar and formation of TLOs at mucosal sites.16Hopken U.E. Wengner A.M. Loddenkemper C. Stein H. Heimesaat M.M. Rehm A. Lipp M. CCR7 deficiency causes ectopic lymphoid neogenesis and disturbed mucosal tissue integrity.Blood. 2007; 109: 886-895Crossref PubMed Scopus (55) Google Scholar, 18Forster R. Davalos-Misslitz A.C. Rot A. CCR7 and its ligands: balancing immunity and tolerance.Nat Rev Immunol. 2008; 8: 362-371Crossref PubMed Scopus (949) Google Scholar Our results show that AIG in CCR7−/− mice occurred as early as 4 weeks of age characterized by lymphocytic mucosal infiltrates, profound loss of parietal cells, increase in the population of mucous cells, and the occurrence of circulating autoantibodies to gastric antigen. AIG manifestation in these mice was associated with the development of gastric TLOs. We focused on the requirement of TLOs and lymph nodes (LNs) for the pathogenesis of spontaneous AIG. We show that local accumulation of autoreactive T lymphocytes promoted the development of spontaneous AIG in CCR7-deficient mice, a process that was independent from the occurrence of TLOs or classic peripheral LNs. C57BL/6 Ly5.1 (CD45.1) congenic mice, lymphotoxin (Lt) α−/− mice,19De Togni P. Goellner J. Ruddle N.H. Streeter P.R. Fick A. Mariathasan S. Smith S.C. Carlson R. Shornick L.P. Strauss-Schoenberger et al.Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin.Science. 1994; 264: 703-707Crossref PubMed Scopus (874) Google Scholar and Rag2−/− mice were obtained from Jackson Laboratories (Bar Harbor, ME). CCR7−/− were generated as previously described13Forster R. Schubel A. Breitfeld D. Kremmer E. Renner-Muller I. Wolf E. Lipp M. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.Cell. 1999; 99: 23-33Abstract Full Text Full Text PDF PubMed Scopus (1917) Google Scholar and backcrossed onto a C57BL/6 background for 12 generations. CCR7−/− mice were crossed with Ltα−/− mice to generate double knockout mice. CD11c.DTR transgenic mice were obtained from Natolio Garbi.20Hochweller K. Striegler J. Hammerling G.J. Garbi N. A novel CD11c.DTR transgenic mouse for depletion of dendritic cells reveals their requirement for homeostatic proliferation of natural killer cells. Eur J Immunol. 2008; 38: 2776-2783Google Scholar Animals were housed in a specific pathogen-free facility at the Max-Delbrück-Center for Molecular Medicine, Berlin, Germany. All animal studies were performed according to institutional and Berlin State guidelines (registered under LaGeSo 0184/04 and 0371/05). Mice were fasted overnight and sacrificed, 50 μL of gastric contents was collected, and the pH was determined using pH paper (Whatman; GE Healthcare, Munic, Germany). The following primary antibodies were used: fluorescein isothiocyanate (FITC)–conjugated Armenian hamster-anti-mouse CD3ε; phycoerythrin (PE)-labeled Armenian hamster-anti-mouse CCR5 (CD195); biotinylated Armenian hamster anti-mouse CXCR3 (CD183); FITC-labeled Armenian hamster anti-mouse CD69; PE-conjugated rat anti-mouse CD44; allophycocyanin (APC)-labeled rat anti-mouse CD4, CD8, and CD19; FITC-labeled rat anti-mouse CD86; PE-labeled rat anti-mouse CD23; PE-labeled rat-anti-mouse CD8α; peridinin chlorophyll protein complex (PerCP)-labeled rat anti-mouse CD8α; PerCP-labeled Armenian hamster anti-mouse CD3ε; PE-coupled Armenian hamster anti-mouse CD103 (all from BioLegend, Kithorn, the Netherlands); FITC-labeled rat anti-mouse CD21/CD35, IgD; FITC-labeled mouse anti-mouse I-Ab; PE-labeled rat anti-mouse IgM (all from BD Biosciences, Heidelberg, Germany); FITC-conjugated rat anti-mouse interferon (IFN)-γ and APC-conjugated rat anti-mouse interleukin 17A (IL-17A) (eBioscience, Frankfurt, Germany); and biotinylated Armenian hamster anti-mouse CD11c (Caltag, Hamburg, Germany). Biotinylated primary antibodies were detected with streptavidin conjugates (BioLegend). For immunohistology, the following primary antibodies were used: mouse-anti-proton pump/H,K-adenosine triphosphatase (ATPase) α-subunit (MBL International Corporation, Woburn, MA); purified rat-anti-mouse CD138; FITC-labeled mouse-anti-mouse CD45.1; Pacific blue–labeled mouse-anti-mouse CD45.2 (all from Biolegend); Alexa 488–labeled rat-anti-mouse CD45R/B220; Pacific blue–labeled rat-anti-mouse CD3; biotinylated rat-anti-mouse CD45R/B220; and biotinylated Armenian hamster-anti-mouse CD11c (all from BioLegend). Biotinylated Ulex europaeus agglutinin I (UEAI; Vector Laboratories, Loerrach, Germany) and FITC-labeled Griffonia simplicifolia lectin (Invitrogen, Darmstadt, Germany) were used to identify pit and mucous neck cells, respectively. Data were acquired on a FACSCanto II flow cytometer (BD Biosciences) and analyzed with Flowjo software versions 8.8.2 and 9.2 (TreeStar, Ashland, OR). For Alcian blue staining, paraffin sections were deparaffinized, incubated with Alcian blue solution (pH 2.5) for 30 minutes at room temperature, rinsed in water, dehydrated, and mounted in Entellan (Merck, Darmstadt, Germany). Images were acquired with a Zeiss Axiophot fluorescence microscope with an Axiocam HRc camera and further processed using AxioVision 4.5 software (all from Carl Zeiss, Göttingen, Germany). Minced tissue of murine stomachs was incubated in RPMI 1640 medium and supplemented with 10% fetal calf serum and 6 mg/mL of collagenase NB4 (Serva, Heidelberg, Germany) for 30 minutes at 37°C. Spleen and mesenteric LNs (mLNs) were injected with 2 mg/mL of collagenase NB4, cut into small pieces, and incubated at 37°C for 30 minutes. Single-cell suspensions were applied on a Ficoll density gradient. Mononuclear cells were isolated from the stomach and incubated for 4 hours at 37°C in RPMI 1640 medium containing 10% fetal calf serum in the presence or absence of a cocktail of ionomycin (1.5 μg/mL), phorbol myristate acetate (40 ng/mL), and brefeldin A (20 μg/mL) (Sigma-Aldrich, Munich, Germany). Surface expression of CD3, CD4, and CD8 and intracellular expression of IFN-γ and IL-17A (Fix & Perm cell permeabilization kit; Caltag) were analyzed by flow cytometry. Stomach tissue of 2-month-old mice was cut into pieces and incubated in 1.25 mL of serum-free RPMI 1640 medium containing penicillin and streptomycin for 31 hours at 37°C and 5% CO2. Supernatants were harvested and assayed for secreted IL-1β, tumor necrosis factor (TNF)-α, IFN-γ, IL-6, IL-10, CCL2 (monocyte chemotactic protein 1), and IL-12p70 using the Cytometric Bead Array Mouse Inflammation Kit and FCAP Array Software (all from BD Biosciences) according to the manufacturer's instructions. Cytokine concentration was indicated by the fluorescent intensity and finally normalized to stomach weight. Gastric epithelial tissue from C57BL/6 wild-type (Wt) and CCR7−/− mice and TLOs from stomachs of CCR7−/− mice were laser microdissected using the PixCell IIe LCM System (Arcturus, Mörfelden-Walldorf, Germany). Cryosections of gastric tissue were mounted on diethyl pyrocarbonate–treated slides, stained, and dehydrated using the HistoGene LCM frozen section staining kit (Arcturus). RNA was isolated using the PicoPure RNA Isolation Kit, followed by two-round amplification of RNA using the RiboAmp Plus Kit (both from Arcturus). For gene expression analysis, antisense RNA was reverse transcribed to cDNA and quantitative PCR was performed in an array format (SABiosciences, Qiagen GmbH, Hilden, Germany). Stomach tissue lysates were prepared in lysis buffer (100 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA-Na2, 1 mmol/L sodium orthovanadate, 1 mmol/L NaF, 1% Triton X-100, and protease inhibitors), and 30 μg of protein was separated by SDS-PAGE. Blots were probed with mouse-anti-proton pump/H,K-ATPase α-subunit antibody (MBL International Corporation) and polyclonal rabbit-anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Abcam), followed by horseradish peroxidase (HRP)–coupled goat anti-mouse or donkey anti-rabbit secondary antibody. Proteins were visualized by chemiluminescence (ECL kit; Thermo Scientific, Bonn, Germany). Densitometric quantification of blot bands was performed using the ImageJ software version 1.42q (NIH). Bone marrow (BM) from 6- to 12-week-old Wt and CCR7−/− mice was transplanted into 8- to 12-week-old, sex-matched Rag2−/− mice. Recipients were sublethally irradiated (7 grays) and reconstituted with 2 to 3 × 107 mixed BM cells (1:1) from Wt C57BL/6/CD45.1 and CCR7−/−C57BL/6/CD45.2 donor mice. Microtiter plates were coated with 10 μg/mL of whole Wt stomach tissue lysate and incubated with sera at a dilution of 1:50. Bound IgG antibodies were detected using HRP-conjugated rabbit anti-mouse antiserum (1:4000, Southern Biotechnology, Aachen, Germany) in conjunction with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (BD). To quantitate serum IL-17 levels, microtiter plates were coated with rat anti-mouse IL-17A (BioLegend). Serum samples were diluted 1:30 and recombinant mouse IL-17A served as a standard. Bound cytokines were detected with biotinylated rat-anti-mouse IL-17A (BioLegend), followed by HRP-conjugated streptavidin and TMB substrate. CCR7−/− mice were injected weekly with 50 to 100 μg of CD4+ cell-depleting antibody GK1.5 antibody (BioLegend) or isotype control antibody (rat IgG2b, κ) starting at 2 weeks of age. CD11c.DTR mice, in which the diphtheria toxin receptor (DTR) is under the control of the CD11c promoter, were used to deplete DCs.20Hochweller K. Striegler J. Hammerling G.J. Garbi N. A novel CD11c.DTR transgenic mouse for depletion of dendritic cells reveals their requirement for homeostatic proliferation of natural killer cells. Eur J Immunol. 2008; 38: 2776-2783Google Scholar Eight DT (8 ng/g of body weight) i.p. injections in CCR7−/− CD11c.DTR transgenic mice every second day induced the depletion of CD11c+ DCs. Results are expressed as arithmetic mean ± SD or SEM, as indicated. P < 0.05 was considered statistically significant and determined by either the Mann-Whitney U-test (one-tailed or two-tailed) or an unpaired, two-tailed Student's t-test, as indicated. We previously reported that the gastrointestinal mucosal tissue of CCR7−/− mice is highly permissive for the formation of TLOs. In aged mice, histologic analysis revealed enlarged stomachs with profound cystic hyperplasia and an increased rate of mucosal proliferation.16Hopken U.E. Wengner A.M. Loddenkemper C. Stein H. Heimesaat M.M. Rehm A. Lipp M. CCR7 deficiency causes ectopic lymphoid neogenesis and disturbed mucosal tissue integrity.Blood. 2007; 109: 886-895Crossref PubMed Scopus (55) Google Scholar Here, immunohistologic analysis of 1- to 12-month-old CCR7−/− mice showed that all animals developed distinct histologic and immunohistologic features characteristic of an AIG. Staining for H+/K+-ATPase α, a marker for parietal cells, revealed a selective loss of parietal cells in CCR7−/− mice (Figure 1A). Atrophy of parietal cells was accompanied by profound lymphoid neogenesis (Figure 1B) exhibiting GC formation in gastric TLOs of aged mice (see Supplemental Figure S1A at http://ajp.amjpathol.org). The incidence of AIG manifestation was more than 95% in all age groups of CCR7−/− mice (1 month, n = 5; 2 months, n = 13; 10 to 16 months, n = 6), whereas Wt mice did not develop any signs of AIG up to 12 months of age. Parietal cell loss was quantitated by immunoblot analysis showing significantly less protein expression for parietal cell antigen H+/K+-ATPase α in whole stomach tissue lysates derived from CCR7−/− compared with Wt mice (Figure 2, A and B).Figure 2Parietal cell loss in CCR7−/− mice is associated with decreased gastric Shh mRNA levels, hypochlorhydria, and increased levels of IL-1β. A: Immunoblotting for parietal H+/K+-ATPase α (HKα) and GAPDH in stomach tissue lysates of 11- to 23-month-old Wt (n = 8; lane 1–8) and CCR7−/− (n = 8; lane 9–16) mice. B: Quantitation of H+/K+-ATPase α protein expression normalized to GAPDH is shown. C: Quantitative PCR for Shh mRNA transcript in stomachs of 2-month-old Wt (n = 6) and CCR7−/− (n = 6) mice. Expression of Shh mRNA was normalized to GAPDH and is depicted as relative gastric Shh mRNA expression in CCR7−/− compared with Wt (set arbitrarily to 1) mice. D: Measurement of intragastric pH in 11- to 23-month-old Wt (n = 7) and CCR7−/− (n = 6) mice using pH indicator paper. E: Determination of IL-1β secretion from stomachs of 2-month-old Wt (n = 5) and CCR7−/− (n = 5) mice. Stomach-derived cells were cultured for 31 hours in RPMI 1640 medium without serum at 37°C. IL-1β release was measured in supernatants by cytokine bead array. Bars represent means ± SD (B and D) or SEM (C and E). *P < 0.05, **P < 0.01; Mann-Whitney U-test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Sonic hedgehog (Shh) is abundantly expressed in the gastric fundic glands, and signaling by Shh protein plays an important role in the maintenance of gastric fund gland homeostasis.21van den Brink G.R. Hardwick J.C. Nielsen C. Xu C. ten Kate F.J. Glickman J. van Deventer S.J. Roberts D.J. Peppelenbosch M.P. Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract.Gut. 2002; 51: 628-633Crossref PubMed Scopus (143) Google Scholar, 22van den Brink G.R. Hardwick J.C. Tytgat G.N. Brink M.A. Ten Kate F.J. Van Deventer S.J. Peppelenbosch M.P. Sonic hedgehog regulates gastric gland morphogenesis in man and mouse.Gastroenterology. 2001; 121: 317-328Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar Because it was described that parietal cell loss is correlated with reduced expression levels of Shh,23Xiao C. Ogle S.A. Schumacher M.A. Orr-Asman M.A. Miller M.L. Lertkowit N. Varro A. Hollande F. Zavros Y. Loss of parietal cell expression of Sonic hedgehog induces hypergastrinemia and hyperproliferation of surface mucous cells.Gastroenterology. 2010; 138 (561.e551-558): 550-561Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 24Waghray M. Zavros Y. Saqui-Salces M. El-Zaatari M. Alamelumangapuram C.B. Todisco A. Eaton K.A. Merchant J.L. Interleukin-1beta promotes gastric atrophy through suppression of Sonic Hedgehog.Gastroenterology. 2010; 138 (572.e561-562): 562-572Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar we quantitated Shh expression in CCR7−/− mice. CCR7−/− mice exhibited decreased gastric Shh gene expression levels (Figure 2C) and hypochlorhydria (Figure 2D) compared with Wt controls. The inflammatory cytokine IL-1β can inhibit parietal cell acid secretion by promoting gastric atrophy through suppression of Shh.24Waghray M. Zavros Y. Saqui-Salces M. El-Zaatari M. Alamelumangapuram C.B. Todisco A. Eaton K.A. Merchant J.L. Interleukin-1beta promotes gastric atrophy through suppression of Sonic Hedgehog.Gastroenterology. 2010; 138 (572.e561-562): 562-572Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Thus, we analyzed whether local gastric IL-1β levels might be altered in CCR7−/− mice. CCR7−/− mice revealed a significant increase in gastric IL-1β production compared with Wt mice (Figure 2E), suggesting that enhanced IL-1β expression induces down-regulation of Shh. Gastric secretion of additional proinflammatory cytokines, ie, IL-6, IL-10, TNF-α, CCL2 (monocyte chemotactic protein 1), and IL-12p70, was not enhanced in CCR7−/− mice (data not shown). Loss of gastric glandular cells frequently leads to a severe alteration of the normal gastric pit architecture, most likely through replacement by intestinal or fibrous tissues.25Marshall A.C. Alderuccio F. Murphy K. Toh B.H. Mechanisms of gastric mucosal cell loss in autoimmune gastritis.Int Rev Immunol. 2005; 24: 123-134Crossref PubMed Scopus (7) Google Scholar Here, stomach sections of 2-month-old Wt or knockout mice were stained for CD138 expression, a marker for gastric pit (foveolar epithelial) cells. The CD138+ cell population was markedly increased in CCR7−/− compared with Wt mice (Figure 3A). In addition, single CD138+ plasma cells were frequently detected within the lamina propria of CCR7−/− mice (Figure 3A, right panel). To further differentiate between pit cells and mucous neck cells, lectin staining was performed. Pit cells (UEAI+) lined the gastric surface and were present along pit walls, whereas mucous neck cells (GSII+) marked the neck region of the gastric gland in Wt mice (Figure 3B, left panel). In sharp contrast, surface pit cells in CCR7−/− mice were found in the vicinity of mucous neck cells and both cell lineages were markedly expanded (Figure 3B, middle and right panels) compared with Wt mice. Next, we stained stomach sections of CCR7−/− mice and Wt controls (1 to 12 months old) for acidic mucopolysaccharides (Figure 3C). Extended Alcian blue staining in young and elder CCR7−/− mice was indicative of an expansion of acidic mucous-secreting cells, demonstrating metaplasia in CCR7−/− mice. In conclusion, CCR7−/− mice exhibited a disturbed gastric fundic gland homeostasis with severely impaired differentiation of mucous-producing cells. Lymphocytes that infiltrate the GM can contribute to epithelial cell destruction and to gastric atrophy during chronic gastritis.26van Driel I.R. Read S. Zwar T.D. Gleeson P.A. Shaping the T cell repertoire to a bona fide autoantigen: lessons from autoimmune gastritis.Curr Opin Immunol. 2005; 17: 570-576Crossref PubMed Scopus (28) Google Scholar To determine the transcriptional profile of the gastric ectopic lymphoid aggregates in CCR7−/− mice versus Wt GM tissue, we microdissected the gastric TLOs and GM tissue of 4- and 12-month-old CCR7−/− and Wt mice, respectively. Quantitative reverse transcription–PCR array analysis focused on genes that were informative on immunoregulatory and proliferative processes. Within gastric TLOs, we
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