Essential Role for Macrophage Migration Inhibitory Factor in Gastritis Induced by Helicobacter pylori
2009; Elsevier BV; Volume: 174; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.080708
ISSN1525-2191
AutoresBenny L.W. Wong, Senlin Zhu, Xiao Ru Huang, Juan Ma, Harry H.X. Xia, Richard Bucala, Benjamin C.Y. Wong, Hui Y. Lan,
Tópico(s)Gastroesophageal reflux and treatments
ResumoMacrophage migration inhibitory factor (MIF) is an upstream regulator of immune and inflammatory responses; however, its role in Helicobacter pylori (HP)-associated gastritis remains unknown. We infected MIF knockout (KO) and wild-type mice with SS1 HP and found that 2 weeks after infection, MIF and its receptor CD74 were markedly up-regulated in wild-type mice. This up-regulation preceded the up-regulation of both tumor necrosis factor-α and intercellular adhesion molecule-1, as well as the development of moderate gastritis at 8 weeks, as determined by a significant infiltration of neutrophils, T cells, and macrophages. In contrast, KO mice were protected against HP-induced gastritis by preventing the up-regulation of CD74 and Th1-mediated immune injury, including a reduction in the Th1 transcriptional factor T-bet and the expression of interferon-γ. Additionally, inhibition of skin delayed type hypersensitivity reactions to HP antigens in KO mice also suggested a critical role for MIF in cell-mediated injury. A regulatory role for MIF in Th1-immune responses was further demonstrated by the finding that antigen-primed CD4+ T cells lacking MIF failed to differentiate into the Th1 phenotype; these cells were instead promoted to Th2 differentiation after challenge with HP antigen in vitro. Results from this study indicated that inhibition of HP-induced innate immune responses and Th1-mediated immune injury may be the key mechanisms by which KO mice failed to develop gastritis after HP infection. Macrophage migration inhibitory factor (MIF) is an upstream regulator of immune and inflammatory responses; however, its role in Helicobacter pylori (HP)-associated gastritis remains unknown. We infected MIF knockout (KO) and wild-type mice with SS1 HP and found that 2 weeks after infection, MIF and its receptor CD74 were markedly up-regulated in wild-type mice. This up-regulation preceded the up-regulation of both tumor necrosis factor-α and intercellular adhesion molecule-1, as well as the development of moderate gastritis at 8 weeks, as determined by a significant infiltration of neutrophils, T cells, and macrophages. In contrast, KO mice were protected against HP-induced gastritis by preventing the up-regulation of CD74 and Th1-mediated immune injury, including a reduction in the Th1 transcriptional factor T-bet and the expression of interferon-γ. Additionally, inhibition of skin delayed type hypersensitivity reactions to HP antigens in KO mice also suggested a critical role for MIF in cell-mediated injury. A regulatory role for MIF in Th1-immune responses was further demonstrated by the finding that antigen-primed CD4+ T cells lacking MIF failed to differentiate into the Th1 phenotype; these cells were instead promoted to Th2 differentiation after challenge with HP antigen in vitro. Results from this study indicated that inhibition of HP-induced innate immune responses and Th1-mediated immune injury may be the key mechanisms by which KO mice failed to develop gastritis after HP infection. Approximately 50% of the world's population has been infected with Helicobacter pylori (HP). HP infection results in the development of gastritis mediated by both innate and adaptive immune responses, including expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-8, and Th1-dominant cytokines such as interferon-γ (IFN-γ) and IL-12. These mediators act by recruiting neutrophils, macrophages, and T cells to the gastric mucosa, resulting in gastric inflammation.1Wilson KT Crabtree JE Immunology of Helicobacter pylori: insights into the failure of the immune response and perspectives on vaccine studies.Gastroenterology. 2007; 133: 288-308Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 2D'Elios MM Andersen LP Helicobacter pylori inflammation, immunity, and vaccines.Helicobacter. 2007; 12: 15-19Crossref PubMed Scopus (31) Google Scholar Migration inhibitory factor (MIF) is a multifunctional cytokine with an upstream regulatory role in inflammation.3Bernhagen J Calandra T Mitchell RA Martin SB Tracey KJ Voelter W Manogue KR Cerami A Bucala R MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia.Nature. 1993; 365: 756-759Crossref PubMed Scopus (941) Google Scholar MIF, originally thought to be produced by activated T cells,4Calandra T Bernhagen J Mitchell RA Bucala R The macrophage is an important and previously unrecognized source of macrophage migration inhibitory factor.J Exp Med. 1994; 179: 1895-1902Crossref PubMed Scopus (902) Google Scholar now is recognized as a pre-formed cytokine that is stored within the cytoplasm of macrophages and many other cell types within the host.5Calandra T Echtenacher B Roy DL Pugin J Metz CN Hultner LH Heumann D Mannel D Bucala R Glauser MR Protection from septic shock by neutralization of macrophage migration inhibitory factor.Nat Med. 2000; 6: 164-170Crossref PubMed Scopus (695) Google Scholar MIF is released on stimulation by stress, endo- and exotoxins, and infection.6Calandra T Roger T Macrophage migration inhibitory factor: a regulator of innate immunity.Nat Rev Immunol. 2003; 3: 791-800Crossref PubMed Scopus (1364) Google Scholar, 7David JR Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction.Proc Natl Acad Sci USA. 1966; 56: 72-77Crossref PubMed Scopus (1104) Google Scholar Once released, MIF acts as a pro-inflammatory cytokine to induce the expression of other inflammatory cytokines/mediators including IL-1, TNFα, IL-2, IL-6, IL-8, IFN-γ, and inducible nitric oxide synthase, which acts to recruit and activate macrophages and T cells, resulting in inflammation and immune-mediated disease.5Calandra T Echtenacher B Roy DL Pugin J Metz CN Hultner LH Heumann D Mannel D Bucala R Glauser MR Protection from septic shock by neutralization of macrophage migration inhibitory factor.Nat Med. 2000; 6: 164-170Crossref PubMed Scopus (695) Google Scholar, 6Calandra T Roger T Macrophage migration inhibitory factor: a regulator of innate immunity.Nat Rev Immunol. 2003; 3: 791-800Crossref PubMed Scopus (1364) Google Scholar, 7David JR Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction.Proc Natl Acad Sci USA. 1966; 56: 72-77Crossref PubMed Scopus (1104) Google Scholar It now is well established that MIF plays a pathogenic role in many inflammatory and immune diseases, including ulcerative colitis,8Ohkawara T Mitsuyama K Takeda H Asaka M Fujiyama Y Nishihira J Lack of macrophage migration inhibitory factor suppresses innate immune response in murine dextran sulfate sodium-induced colitis.Scand J Gastroenterol. 2008; 29: 1-9Google Scholar, 9Ohkawara T Miyashita K Nishihira J Mitsuyama K Takeda H Kato M Kondo N Yamasaki Y Sata M Yoshiki T Sugiyama T Asaka M Transgenic over-expression of macrophage migration inhibitory factor renders mice markedly more susceptible to experimental colitis.Clin Exp Immunol. 2005; 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146: 2942-2951Crossref PubMed Scopus (110) Google Scholar acute distress syndrome,14Donnelly SC Haslett C Reid PT Grant IS Wallace WA Metz CN Bruce LJ Bucala R Regulatory role for macrophage migration inhibitory factor in acute respiratory distress syndrome.Nat Med. 1997; 3: 320-323Crossref PubMed Scopus (402) Google Scholar rheumatoid arthritis,15Morand EF Leech M Bernhagen J MIF: a new cytokine link between rheumatoid arthritis and atherosclerosis.Nat Rev Drug Discov. 2006; 5: 399-410Crossref PubMed Scopus (299) Google Scholar atherosclerosis,16Bernhagen J Krohn R Lue H Gregory JL Zernecke A Koenen RR Dewor M Georgiev I Schober A Leng L Kooistra T Fingerle-Rowson G Ghezzi P Kleemann R McColl SR Bucala R Hickey MJ Weber C MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment.Nat Med. 2007; 13: 587-596Crossref PubMed Scopus (959) Google Scholar and kidney diseases.17Lan HY Bacher M Yang N Mu W Nikolic-Paterson DJ Metz C Meinhardt A Bucala R Atkins RC The pathogenic role of macrophage migration inhibitory factor in immunologically induced kidney disease in the rat.J Exp Med. 1997; 185: 1455-1465Crossref PubMed Scopus (257) Google Scholar Evidence to support the importance of MIF in gastric inflammation comes from our recent studies that MIF is up-regulated in gastric inflammation and plays a role in gastric ulcer induced by acetic acid.18Huang XR Chun Hui CW Chen YX Wong BC Fung PC Metz C Cho CH Hui WM Bucala R Lam SK Lan HY Macrophage migration inhibitory factor is an important mediator in the pathogenesis of gastric inflammation in rats.Gastroenterology. 2001; 121: 619-630Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Furthermore, we have also shown that up-regulation of gastric MIF is associated with HP infection.19Xia HH Lam SK Huang XR Wong WM Leung SY Yuen ST Lan HY Wong BC Helicobacter pylori infection is associated with increased expression of macrophage migratory inhibitory factor–by epithelial cells, T cells, and macrophages–in gastric mucosa.J Infect Dis. 2004; 190: 293-302Crossref PubMed Scopus (42) Google Scholar, 20He XX Yang J Ding YW Liu W Shen QY Xia HH Increased epithelial and serum expression of macrophage migration inhibitory factor (MIF) in gastric cancer: potential role of MIF in gastric carcinogenesis.Gut. 2006; 55: 797-802Crossref PubMed Scopus (97) Google Scholar Interestingly, the association between functional promoter polymorphisms in the MIF gene and the progression of gastric mucosal inflammation and the development of mucosal atrophy suggests that MIF also is a risk factor for the subsequent development of gastric cancer.21Arisawa T Tahara T Shibata T Nagasaka M Nakamura M Kamiya Y Fujita H Yoshioka D Arima Y Okubo M Hirata I Nakano H De la Cruz V Functional promoter polymorphisms of the macrophage migration inhibitory factor gene in gastric carcinogenesis.Oncol Rep. 2008; 19: 223-228PubMed Google Scholar Nevertheless, the exact pathogenic role of MIF in HP-induced gastritis remains unclear. Emerging evidence from recent studies demonstrates that Th1-mediated immune mechanisms may be critical in the development of gastritis associated with HP infection, in both animal models and human patients.22Eaton KA Mefford M Thevenot T The role of T cell subsets and cytokines in the pathogenesis of Helicobacter pylori gastritis in mice.J Immunol. 2001; 166: 7456-7461Crossref PubMed Scopus (245) Google Scholar, 23Taylor JM Ziman ME Huff JL Moroski NM Vajdy M Solnick JV Helicobacter pylori lipopolysaccharide promotes a Th1 type immune response in immunized mice.Vaccine. 2006; 24: 4987-4994Crossref PubMed Scopus (38) Google Scholar, 24Smythies LE Waites KB Lindsey JR Harris PR Ghiara P Smith PD Helicobacter pylori-induced mucosal inflammation is Th1 mediated and exacerbated in IL-4, but not IFN-gamma, gene-deficient mice.J Immunol. 2000; 165: 1022-1029Crossref PubMed Scopus (311) Google Scholar, 25Akhiani AA Pappo J Kabok Z Schön K Gao W Franzén LE Lycke N Protection against Helicobacter pylori infection following immunization is IL-12-dependent and mediated by Th1 cells.J Immunol. 2002; 169: 6977-6984Crossref PubMed Scopus (189) Google Scholar, 26Bamford KB Fan X Crowe SE Leary JF Gourley WK Luthra GK Brooks EG Graham DY Reyes VE Ernst PB 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 (501) Google Scholar, 27Eaton KA Benson LH Haeger J Gray BM Role of transcription factor T-bet expression by CD4+ cells in gastritis due to Helicobacter pylori in mice.Infect Immun. 2006; 74: 4673-4684Crossref PubMed Scopus (37) Google Scholar It has been long considered that MIF is a T cell cytokine and mediates Th1 immune response.28Bernhagen J Bacher M Calandra T Metz CN Doty SB Donnelly T Bucala R An essential role for macrophage migration inhibitory factor in the tuberculin delayed-type hypersensitivity reaction.J Exp Med. 1996; 183: 277-282Crossref PubMed Scopus (253) Google Scholar Thus, we hypothesized that MIF may be a critical mediator in regulating Th1-mediated gastritis in response to HP infection. In the present study, we tested this hypothesis and explored the pathogenic role of MIF in HP-induced gastritis in MIF knockout (MIF KO) mice. In addition, the mechanism of MIF in regulating HP-induced Th1 and Th2 phenotype differentiation was studied in vitro in HP-antigen-sensitized CD4+ T cells. H. pylori, Sydney Strain 1(SS1) was grown on Brucella Agar (Oxoid, UK) supplemented with 8% horse blood and selective supplement for H. pylori (Oxoid) for 3 days under 37°C before harvest. Lactobacilli rhamnosus was obtained from American Tissue Culture Collection (ATCC 7469). The bacteria were grown on Malt Extract Agar (Oxoid) at 37°C for 2 days. A single colony was selected and then cultured in the lactic broth (Oxoid, UK) under 37°C for 1 day with aerobic conditions created by shaking vigorously (300 rpm). A standard curve of OD600 versus colony forming unit was used to give a quantity of the bacteria in the suspension. MIF wild-type and MIF KO mice (C57/BL6, age 8 weeks, 20 g) were generated as previously described.29Fingerle-Rowson G Petrenko O Metz CN Forsthuber TG Mitchell R Huss R Moll U Müller W Bucala R The p53-dependent effects of macrophage migration inhibitory factor revealed by gene targeting.Proc Natl Acad Sci USA. 2003; 100: 9354-9359Crossref PubMed Scopus (250) Google Scholar All mice were housed in the Animal Unit at the University of Hong Kong with free access to food and water. A mouse model of gastritis was induced in groups of six MIF wild-type and KO mice by intragastric injection of HP (SS1, 1 × 107 colony forming units). HP suspended in 500 μl Brucella broth (Oxoid) was administered via gavage tube for three times every other day in 1 week. Based on our pilot experiments, the use of this dose produced a moderate gastritis in mouse antrum of the stomach. Groups of six MIF wild-type or KO mice were euthanized at 2 weeks and 8 weeks after the last HP inoculation. Gastric tissues including the body and antrum of stomach were collected and fixed in methyl Carnoy's solution for 24 hours for histology and immunohistochemistry or snap-frozen and stored at −80°C for RNA extraction. In addition, groups of six normal MIF wild-type and KO mice were used as controls. All experimental procedures were approved by the Hong Kong Hygiene Department and the Committee on the Use of Live Animals for Teaching and Research at the University of Hong Kong. Mice were sensitized by injection of HP sonicated protein (500 μg/ml) mixed with the complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO) in the ratio of 1:1/vol:vol by sonication. The mixture was injected subcutaneously into both flanks of the mice (50 μl/each site). Seven days after immunization, skin delayed type hypersensitivity was tested and splenocytes were isolated for culture as described below. Seven days after immunization as described above, skin delayed-type hypersensitivity (DTH) to the HP antigen was tested by subcutaneous injection of 2 μg of HP sonicated protein into the left footpad of the mouse. Two μg of sonicated protein from L. rhamnosus was injected into the right footpad of the mouse as a negative control. Skin swelling was measured by electronic caliper (Gilson, MO) after 24 hours and tissues were fixed with methyl Carnoy's solution for 24 hours and embedded in paraffin for immunohistochemical examination. Mouse splenocytes were isolated by a sieving method at day 7 after immunization with the HP antigen. After washing, single spleen cells at 2 × 106 were seeded on the 24-well plate in Dulbecco's Modified Eagle medium supplemented with 20% fetal bovine serum, 0.1% penicillin, and streptomycin. Cells were stimulated with HP or L. rhamnosus protein at concentrations 0, 5, 10, 25, and 50 μg/ml for 0, 48, and 72 hours. After stimulation, the differentiation of Th1 (CD4+IFN-γ+) or Th2 (CD4+IL-4+) T cells was quantitatively analyzed by two-color flow cytometry as described below. Changes in gastric histology were examined in methyl Carnoy's- fixed, paraffin -embedded tissue sections (4 μm) stained with hematoxylin and eosin or periodic acid-Schiff. Histological changes were scored by updated Sydney System.30Dixon MF Genta RM Yardley JH Correa P Classification and grading of gastritis. The updated Sydney System International Workshop on the histopathology of gastritis, Houston 1994.Am J Surg Pathol. 1996; 20: 1161-1181Crossref PubMed Scopus (4567) Google Scholar Neutrophils infiltrating the gastric mucosa were identified by their polymorph nuclear morphology and the number of polymorph nuclear cells in the entire antrum was counted under high-power fields (×40) by means of a 0.025-mm2 graticule fitted in the eyepiece of the microscope. Data obtained were expressed as cells per mm2. For immunohistochemistry, sections (4 μm) were stained for over night at 4°C with the primary antibodies, including the goat anti-CD3 (DAKO Corporation, Carpinteria, CA), rat anti-F4/80 (macrophage marker (CD68); Serotec, Raleigh, NC), goat anti-TNF-α, goat anti-IFN-γ, goat anti-HP, rabbit anti-CD74 and MIF (all from Santa Cruz Biotechnology, CA) using the microwave antigen retrieval technique as previously described.18Huang XR Chun Hui CW Chen YX Wong BC Fung PC Metz C Cho CH Hui WM Bucala R Lam SK Lan HY Macrophage migration inhibitory factor is an important mediator in the pathogenesis of gastric inflammation in rats.Gastroenterology. 2001; 121: 619-630Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 31Lan HY Mu W Nikolic-Paterson DJ Atkins RC A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: use of microwave oven heating to block antibody crossreactivity and retrieve antigens.J Histochem Cytochem. 2005; 43: 97-102Crossref Scopus (349) Google Scholar After washing, sections were stained with the secondary antibodies with peroxidase-conjugated anti-rabbit or goat antibody followed by goat or rabbit peroxidase anti-peroxidase (Dako Corporation, CA) for 1 hour at room temperature. After being washed, color was developed with 3, 3-diaminobenzidine and counterstained with hematoxylin. An isotype-matched IgG (Sigma) was used as a negative control throughout the study. Quantitative analysis of immunostaining was performed on coded slides as described previously.18Huang XR Chun Hui CW Chen YX Wong BC Fung PC Metz C Cho CH Hui WM Bucala R Lam SK Lan HY Macrophage migration inhibitory factor is an important mediator in the pathogenesis of gastric inflammation in rats.Gastroenterology. 2001; 121: 619-630Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Briefly, the number of positive cells for CD3 and F4/80 was counted under high-power fields (×40) by means of a 0.025-mm2 graticule fitted in the eyepiece of the microscope. The entire antrum and area of DTH-tested skin were examined. Positive cells were expressed as cells per mm2. Expression of gastric MIF, CD74, IFN-γ, and TNFα in the entire antrum was determined using the quantitative Image Analysis System (Carl Zesis Microimaging, Thornwood, NY). Briefly, the examined area of antrum was outlined, the positive staining patterns were identified, and the percent positive area calculated. Data were expressed as percent positive area examined. The expression of genes of interest within the tissues was detected by quantitative real-time reverse transcription-PCR Total RNA was extracted from the gastric tissues or cultured spleen cells by RNA extraction kit and dissolved in RNAase free water. cDNA was prepared from RNA using the Perkin-Elmer Reverse Transcription System (Perkin Elmer Inc., Norwalk, CT) as per manufacturer instruction. Real-time PCR was run with the Opticon real time PCR machine (MJ Research Inc., Waltham, MA) using IQ SYBR green supermix reagent (Bio-Rad, Herculus, CA). The specificity of the real time PCR was confirmed via routine agarose gel electrophoresis and Melting-curve analysis. The gene for glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard. The primers used in this study were: MIF: 5′-GCAAGCCCGCACAGTACA-3′, reverse 5′-CGTTCGTGCCGCTAAAAGTC-3′; CD74: 5′-TTCCGAAATCTGCCAAACCT-3′, reverse 5′-GACATTGGACGCATCAGCAA-3′; IFN-γ: forward 5′-TGGATATCTGGAGGAACTGGCA-3′, reverse 5′-TTTCGCCTTGCTGTTGCTGA-3′; T-bet: forward 5′-AAGCAAGGACGGCGAATGTT-3′, reverse 5′-TCCACCAAGACCACATCCACAA-3′; TNFα: 5′-CATGAGCACAGAAAGCATGATCCG-3′, reverse 5′-AAGCAGGAATGAGAAGAGGCTGAG-3′; ICAM-1: forward 5′-TCAGGTATCCATCCATCCCAGAGA-3′, reverse 5′-AGCTCATCTTTCAGCCACTGAGTC-3′; and glyceraldehyde-3-phosphate dehydrogenase: forward 5′-TGCTGAGTATGTCGTGGAGTCTA-3′, reverse 5′-AGTGGGAGTTTGCTGTTGAAATC-3′. After culture, splenocytes were collected and fixed for 30 minutes in 2% paraformaldehyde and permeablized by 0.5% saponin solution for 5 hours under 4°C. Then cells were stained with phycoerythrin-conjugated rat anti-mouse CD4 (e-Bioscience) for 30 minutes at 4°C, followed by a rabbit anti-mouse IFN-γ-fluorescein isothiocyanate (FITC) (Serotec) or IL-4-FITC (e-Bioscience). After being extensively washed, single cells were analyzed by flow cytometry (Cytomic FC500MPL, Beckman Coulter, High Wycombe, UK). Data obtained from both in vivo and in vitro studies were expressed as the mean ± SEM. Statistical analyses were performed using one-way analysis of variance, followed by the t-test from GraphPad Prism 3.0 (GraphPad Software, Inc. San Diego, CA). Both MIF KO and wild-type mice infected with HP exhibited strong positivity for the rapid urease test. Immunohistochemically, both MIF KO and wild-type mouse stomach at 8 weeks after HP infection showed equivalent HP positive immunostaining with the anti-HP antibody (Figure 1A), indicating that mice lacking MIF did not influence the extent of HP infection. Histologically, periodic acid-Schiff-stained sections showed that although there was not detectable abnormalities in gastric histology in all animals at 2 weeks after HP infection, a moderate gastritis with many polymorph nuclear cells (neutrophils) and mononuclear cells infiltrating the mucosa was developed in MIF wild-type mice at 8 weeks after HP infection, which was absent in mice lacking MIF (Figure 1B). This was further confirmed by histological score and the influx of neutrophils (Figure 1, C and D). We next examined whether HP infection up-regulated MIF and its receptor CD74 in murine gastric tissue. In uninfected gastric tissue, immunohistochemistry and quantitative real-time PCR showed minimal MIF mRNA and protein expression was constitutively expressed by gastric epithelial cells in wild-type, but not in KO mice (Figure 2, Figure 3). The MIF receptor, CD74, was weakly expressed in the gastric tissues in both normal wild-type and KO mice (Figure 2, Figure 3). By contrast, both real-time PCR and immunohistochemistry also showed that a marked up-regulation of gastric MIF and CD74 mRNA and protein was found in the wild-type mice at 2 weeks after HP infection, preceding the development of gastric inflammation at 8 weeks as shown histologically in Figure 1, B–D. A further increase in MIF and CD74 in MIF wild-type mice was detected at 8 weeks after HP infection (Figure 2, Figure 3). However, mice lacking MIF remained negative for MIF and did not show an increase in CD74 mRNA and protein expression in HP-infected stomach through out the entire disease course (Figure 2, Figure 3).Figure 3Real-time PCR shows mRNA expression of MIF, CD 74, TNFα, and ICAM-1 in gastric tissues in MIF wild-type (WT) and KO mice with or without HP infection. A: MIF mRNA expression at 8 weeks after HP infection. B and C: CD74 mRNA expression at 2 and 8 weeks after HP infection. D and E: TNFα mRNA expression at 2 and 8 weeks after HP infection. F: Icam-1 expression at 8 weeks after HP infection. Each bar represents mean ± SEM for a group of six mice. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with wild-type mice without HP infection; #P < 0.05, ##P < 0.01 as compared with the time-matched wild-type mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunohistochemistry revealed that there were few CD3+ T cells and F4/80+ macrophages in normal gastric mucosa in both MIF KO and wild-type mice (Figure 4, Aa, b, Ba, b, C, and D). At 2 weeks after HP infection, although gastric F4/80+ macrophages remained low, CD3+ T cells was significantly increased in wild-type mice (Figure 4). By 8 weeks after HP infection, all MIF wild-type mice exhibited a prominent macrophage and T cell infiltration with the development of moderate gastritis (Figure 4). By contrast, there was no increase in macrophage and T cell infiltration in the gastric tissues of MIF KO though out the disease course after HP-infection, indicating that MIF deficiency resulted in protection from gastric inflammation. It has been well demonstrated that the pro-inflammatory cytokine TNFα and adhesion molecule ICAM-1 play a critical role in gastric inflammation associated with HP infection.1Wilson KT Crabtree JE Immunology of Helicobacter pylori: insights into the failure of the immune response and perspectives on vaccine studies.Gastroenterology. 2007; 133: 288-308Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 2D'Elios MM Andersen LP Helicobacter pylori inflammation, immunity, and vaccines.Helicobacter. 2007; 12: 15-19Crossref PubMed Scopus (31) Google Scholar We examined gastric TNFα and ICAM-1 expression by immunohistochemistry and real-time PCR and observed marked up-regulation of gastric TNFα and ICAM-1 in wild-type mice at 8 weeks after HP infection (Figure 3, Figure 5). By contrast, mice lacking MIF exhibited normal levels of TNFα and ICAM-1 expression (Figure 3, Figure 5). Th1-mediated immune injury is critical for the development of gastritis associated with HP infection22Eaton KA Mefford M Thevenot T The role of T cell subsets and cytokines in the pathogenesis of Helicobacter pylori gastritis in mice.J Immunol. 2001; 166: 7456-7461Crossref PubMed Scopus (245) Google Scholar, 23Taylor JM Ziman ME Huff JL Moroski NM Vajdy M Solnick JV Helicobacter pylori lipopolysaccharide promotes a Th1 type immune response in immunized mice.Vaccine. 2006; 24: 4987-4994Crossref PubMed Scopus (38) Google Scholar, 24Smythies LE Waites KB Lindsey JR Harris PR Ghiara P Smith PD Helicobacter pylori-induced mucosal inflammation is Th1 mediated and exacerbated in IL-4, but not IFN-gamma, gene-deficient mice.J Immunol. 2000; 165: 1022-1029Crossref PubMed Scopus (311) Google Scholar, 25Akhiani AA Pappo J Kabok Z Schön K Gao W Franzén LE Lycke N Protection against Helicobacter pylori infection following immunization is IL-12-dependent and mediated by Th1 cells.J Immunol. 2002; 169: 6977-6984Crossref PubMed Scopus (189) Google Scholar, 26Bamford KB Fan X Crowe SE Leary JF Gourley WK Luthra GK Brooks EG Graham DY Reyes VE Ernst PB 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 (501) Google Scholar, 27Eaton KA Benson LH Haeger J Gray BM Role of transcription factor T-bet expression by CD4+ cells in gastritis due to Helicobacter pylori in mice.Infect Immun. 2006; 74: 4673-4684Crossref PubMed Scopus (37) Google Scholar and MIF has been reported to be associated with a Th1-type T cell response.7David JR Delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction.Proc Natl Acad Sci USA. 1966; 56: 72-77Crossref PubMed Scopus (1104) Google Scholar, 28Bernhagen J Bacher M Calandra T Metz CN Doty SB Donnelly T Bucala R An essential role for macrophage migration inhibitory factor in the tuberculin delayed-type hypersensitivity reaction.J Exp Med. 1996; 183: 277-282Crossref PubMed Scopus (253) Google Scholar Accordingly, we examined the pathogenic role of MIF in Th1-mediated immune response in gastric tissues after HP infection. As shown in Figure 6C, a Th1 master transcriptional factor T-bet mRNA was significantly up-regulated in MIF wild-type mice at 2 weeks after HP infection, contributing significantly to a marked up-regulation of a Th1 signature cytokine IFN-γ at 8 weeks after infection with HP (Figure 5, Figure 6). By contrast, mice lacking MIF exhibited normal mRNA and protein levels of IFN-γ and T-bet in gastric tissues infected with HP through out the entire disease ti
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