Unique Role of Junctional Adhesion Molecule-A in Maintaining Mucosal Homeostasis in Inflammatory Bowel Disease
2008; Elsevier BV; Volume: 135; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2008.04.002
ISSN1528-0012
AutoresStefania Vetrano, María Rescigno, Maria Rosaria, Carmen Correale, Cristiano Rumio, Andrea Doni, Massimo Claudio Fantini, Andreas Sturm, Elena Monica Borroni, Alessandro Repici, Massimo Locati, Alberto Malesci, Elisabetta Dejana, Silvio Danese,
Tópico(s)Immune Response and Inflammation
ResumoBackground & Aims: Junctional adhesion molecule-A (JAM-A) is localized at the tight junctions and controls leukocyte migration into the tissues. However, its functional role in inflammatory bowel disease (IBD) is unexplored. Methods: Control, Crohn's disease (CD), and ulcerative colitis (UC) tissue specimens were studied for JAM-A expression, as well as the colon of mice given dextran sodium sulfate (DSS). Wild-type and JAM-A-/-, Tie-2-Cre-JAM-A-/- (endothelial/hematopoietic-specific JAM inactivation) mice were studied for susceptibility to DSS. Disease activity and colonic inflammation were assessed using a disease activity index histology and endoscopy, and mucosal cytokines were measured by enzyme-linked immunosorbent assay. JAM-A function was investigated by RNA silencing in epithelial cells, and apoptosis was measured. Results: In both CD and UC, as well as in experimental colitis, there is a loss of epithelial but not endothelial JAM-A expression. Deletion of JAM-A results in a dramatic increase in susceptibility to DSS colitis, as assessed by weight loss, disease activity index, histologic and endoscopic severity, and strikingly high mortality rates. This is not caused by the absence of JAM-A in the endothelial or hematopoietic compartments because Tie-2-Cre–JAM-A-/- mice are no more susceptible to DSS colitis than wild-type animals. JAM-A-/- mice displayed increased intestinal permeability and inflammatory cytokine production, and marked epithelial apoptosis. Silencing of JAM-A in intestinal epithelial cells resulted in increased permeability in vitro. Conclusions: Our results show a nonredundant and novel role of JAM-A in controlling mucosal homeostasis by regulating the integrity and permeability of epithelial barrier function. Background & Aims: Junctional adhesion molecule-A (JAM-A) is localized at the tight junctions and controls leukocyte migration into the tissues. However, its functional role in inflammatory bowel disease (IBD) is unexplored. Methods: Control, Crohn's disease (CD), and ulcerative colitis (UC) tissue specimens were studied for JAM-A expression, as well as the colon of mice given dextran sodium sulfate (DSS). Wild-type and JAM-A-/-, Tie-2-Cre-JAM-A-/- (endothelial/hematopoietic-specific JAM inactivation) mice were studied for susceptibility to DSS. Disease activity and colonic inflammation were assessed using a disease activity index histology and endoscopy, and mucosal cytokines were measured by enzyme-linked immunosorbent assay. JAM-A function was investigated by RNA silencing in epithelial cells, and apoptosis was measured. Results: In both CD and UC, as well as in experimental colitis, there is a loss of epithelial but not endothelial JAM-A expression. Deletion of JAM-A results in a dramatic increase in susceptibility to DSS colitis, as assessed by weight loss, disease activity index, histologic and endoscopic severity, and strikingly high mortality rates. This is not caused by the absence of JAM-A in the endothelial or hematopoietic compartments because Tie-2-Cre–JAM-A-/- mice are no more susceptible to DSS colitis than wild-type animals. JAM-A-/- mice displayed increased intestinal permeability and inflammatory cytokine production, and marked epithelial apoptosis. Silencing of JAM-A in intestinal epithelial cells resulted in increased permeability in vitro. Conclusions: Our results show a nonredundant and novel role of JAM-A in controlling mucosal homeostasis by regulating the integrity and permeability of epithelial barrier function. Junctional adhesion molecule-A (JAM-A) belongs to a family of cell adhesion molecules that localizes at intercellular junctions and mediates several different physiologic processes, such as junction assembly and leukocyte migration.1Nourshargh S. Krombach F. Dejana E. The role of JAM-A and PECAM-1 in modulating leukocyte infiltration in inflamed and ischemic tissues.J Leukoc Biol. 2006; 80: 714-718Crossref PubMed Scopus (115) Google Scholar, 2Naik U.P. Eckfeld K. Junctional adhesion molecule 1 (JAM-1).J Biol Regul Homeost Agents. 2003; 17: 341-347PubMed Google Scholar JAM-A expression is particularly abundant at tight junctions of endothelial and epithelial cells, but also is present on leukocytes.3Utech M. Bruwer M. Nusrat A. Tight junctions and cell-cell interactions.Methods Mol Biol. 2006; 341: 185-195PubMed Google Scholar At the endothelial sites, JAM-A has been shown to be crucially involved in mediating leukocyte transmigration through the paracellular pathway.1Nourshargh S. Krombach F. Dejana E. The role of JAM-A and PECAM-1 in modulating leukocyte infiltration in inflamed and ischemic tissues.J Leukoc Biol. 2006; 80: 714-718Crossref PubMed Scopus (115) Google Scholar, 4Weber C. Fraemohs L. Dejana E. The role of junctional adhesion molecules in vascular inflammation.Nat Rev Immunol. 2007; 7: 467-477Crossref PubMed Scopus (396) Google Scholar Indeed, during vascular inflammation such as in atherosclerosis, JAM-A is up-regulated and controls leukocyte influx into the atherosclerotic lesion.5Ostermann G. Fraemohs L. Baltus T. et al.Involvement of JAM-A in mononuclear cell recruitment on inflamed or atherosclerotic endothelium: inhibition by soluble JAM-A.Arterioscler Thromb Vasc Biol. 2005; 25: 729-735Crossref PubMed Scopus (73) Google Scholar, 6Zernecke A. Liehn E.A. Fraemohs L. et al.Importance of junctional adhesion molecule-A for neointimal lesion formation and infiltration in atherosclerosis-prone mice.Arterioscler Thromb Vasc Biol. 2006; 26: e10-e13Crossref PubMed Scopus (66) Google Scholar In addition, JAM-A blockade by monoclonal antibody or genetic deletion in mice has been proven to reduce leukocyte infiltration in in vivo models of inflammation, such as experimental cytokine-induced meningitis, air-pouch model of skin inflammation, and in ischemia-reperfusion injury models in the liver and heart.7Del Maschio A. De Luigi A. Martin-Padura I. et al.Leukocyte recruitment in the cerebrospinal fluid of mice with experimental meningitis is inhibited by an antibody to junctional adhesion molecule (JAM).J Exp Med. 1999; 190: 1351-1356Crossref PubMed Scopus (254) Google Scholar, 8Cera M.R. Del Prete A. Vecchi A. et al.Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-A-deficient mice.J Clin Invest. 2004; 114: 729-738Crossref PubMed Scopus (145) Google Scholar, 9Corada M. Chimenti S. Cera M.R. et al.Junctional adhesion molecule-A-deficient polymorphonuclear cells show reduced diapedesis in peritonitis and heart ischemia-reperfusion injury.Proc Natl Acad Sci U S A. 2005; 102: 10634-10639Crossref PubMed Scopus (99) Google Scholar, 10Khandoga A. Kessler J.S. Meissner H. et al.Junctional adhesion molecule-A deficiency increases hepatic ischemia-reperfusion injury despite reduction of neutrophil transendothelial migration.Blood. 2005; 106: 725-733Crossref PubMed Scopus (90) Google Scholar At epithelial sites, JAM-A has been reported to regulate epithelial barrier function in the intestine and the cornea.11Bruewer M. Utech M. Ivanov A.I. et al.Interferon-gamma induces internalization of epithelial tight junction proteins via a macropinocytosis-like process.FASEB J. 2005; 19: 923-933Crossref PubMed Scopus (290) Google Scholar, 12Mandell K.J. Holley G.P. Parkos C.A. et al.Antibody blockade of junctional adhesion molecule-A in rabbit corneal endothelial tight junctions produces corneal swelling.Invest Ophthalmol Vis Sci. 2006; 47: 2408-2416Crossref PubMed Scopus (46) Google Scholar, 13Laukoetter M.G. Bruewer M. Nusrat A. Regulation of the intestinal epithelial barrier by the apical junctional complex.Curr Opin Gastroenterol. 2006; 22: 85-89Crossref PubMed Scopus (199) Google Scholar Indeed, JAM-A inhibition affects epithelial barrier recovery after an in vitro model of epithelial injury and plays an important role in epithelial tight junction assembly.13Laukoetter M.G. Bruewer M. Nusrat A. Regulation of the intestinal epithelial barrier by the apical junctional complex.Curr Opin Gastroenterol. 2006; 22: 85-89Crossref PubMed Scopus (199) Google Scholar However, the expression and functional roles in vivo of JAM-A have not been precisely characterized in intestinal inflammation.Crohn's disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD), are chronic intestinal disorders. Although their etiology still is unknown, clear evidence points out that besides the widely recognized aberrant function of the immune cells, nonimmune cells such as those in the epithelium and endothelium actively participate in disease pathogenesis.14Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis.Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1855) Google Scholar, 15Danese S. Fiocchi C. Etiopathogenesis of inflammatory bowel diseases.World J Gastroenterol. 2006; 12: 4807-4812PubMed Google Scholar, 16Danese S. Inflammation and the mucosal microcirculation in inflammatory bowel disease: the ebb and flow.Curr Opin Gastroenterol. 2007; 23: 384-389Crossref PubMed Scopus (50) Google Scholar Many molecules have been recognized as key effectors in both CD and UC pathogenesis, but the functional role of JAM-A in IBD pathogenesis has not been addressed fully.Therefore, the aim of the present study was to analyze the expression of JAM-A in the normal and inflamed intestine, and to investigate the functional role of JAM-A in experimental colitis. Our results show that in both forms of human IBD and in experimental colitis, JAM-A is dramatically down-regulated at epithelial tight junctions. Furthermore, endothelial JAM-A is not involved in the regulation of intestinal inflammation, whereas epithelial JAM-A plays a key role in maintaining intestinal barrier function through the regulation of epithelial cell barrier integrity.Materials and MethodsReagents and AntibodiesAll the tissue culture reagents were purchased from Life Technologies (Paisley, UK). Caco-2 cells were routinely cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 1 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 0.1 mmol/L nonessential amino acids, and 100 U/mL antibiotics (penicillin and streptomycin).Mouse anti-human JAM-A (BV16) and rat anti-mouse JAM-A (BV12 and BV11) monoclonal antibodies were generated as previously reported7Del Maschio A. De Luigi A. Martin-Padura I. et al.Leukocyte recruitment in the cerebrospinal fluid of mice with experimental meningitis is inhibited by an antibody to junctional adhesion molecule (JAM).J Exp Med. 1999; 190: 1351-1356Crossref PubMed Scopus (254) Google Scholar; rabbit anti-occludin and anti–zonula occludens 1 (ZO-1), and rat anti–E-cadherin were from Zymed-Invitrogen (San Francisco, CA); rabbit anti–β-catenin was from AbCam (Science Park Cambridge, UK); secondary Abs 488- or Cy3-conjugated were from Molecular Probes (San Francisco, CA) and Jackson ImmunoResearch Laboratories (Westgrove, PA), respectively. Alexa Fluor 488–conjugated goat antimouse, antirat, or anti rabbit immunoglobulin (Ig)G and/or Alexa Fluor 594–conjugated goat antimouse or antirat IgG antibodies were from Invitrogen. Antibody against murine pan-cytokeratin was from Santa Cruz Biotechnology (Santa Cruz, CA), and anti–claudins-1, -2, and -3 and cingulin were from Zymed.PatientsIntestinal tissues were obtained from surgical specimens of patients with CD, UC, and as control from normal areas of the intestine of patients admitted for bowel resection because of colon cancer, polyps, or diverticulosis. Specimens were frozen in optimum cutting temperature tissue on dry ice and stored at −80°C. Human studies were approved by the ethical committee of the Istituto Clinico Humanitas.AnimalsMale JAM-A+/+, JAM-A-/-, and Tie-2 Cre JAM-A-/- mice, generated and genotyped as described,8Cera M.R. Del Prete A. Vecchi A. et al.Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-A-deficient mice.J Clin Invest. 2004; 114: 729-738Crossref PubMed Scopus (145) Google Scholar were used in this study. These mice were on a C57BL/6J background, and wild-type littermates were used as controls in all experiments. All mice were maintained under pathogen-free conditions. Procedures involving animals and their care conformed to institutional guidelines in agreement with national and international law, after approval by the ethical committee.Induction of ColitisTen- to 12-week-old mice received 2% dextran sodium sulfate (DSS) (molecular mass, 40 kilodaltons; MP Biomedicals, Irvine, CA) ad libitum in filter-purified drinking water for 7 days. Colitis was scored daily using standard parameters that included body weight, diarrhea, presence of blood in the stools, and rectal prolapse, and a disease activity index was calculated as reported.17Danese S. Sans M. Spencer D.M. et al.Angiogenesis blockade as a new therapeutic approach to experimental colitis.Gut. 2007; 56: 855-862Crossref PubMed Scopus (106) Google Scholar, 18Scaldaferri F. Sans M. Vetrano S. et al.Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease.J Clin Invest. 2007; 117: 1951-1960Crossref PubMed Scopus (105) Google Scholar Grading of intestinal inflammation was determined as previously described18Scaldaferri F. Sans M. Vetrano S. et al.Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease.J Clin Invest. 2007; 117: 1951-1960Crossref PubMed Scopus (105) Google Scholar in a blinded fashion: no inflammation was scored as 0; modest numbers of infiltrating cells in the lamina propria was scored as 1; infiltration of mononuclear cells leading to separation of crypts and mild mucosal hyperplasia was scored as 2; massive infiltration with inflammatory cells accompanied by disrupted mucosal architecture, loss of goblet cells, and marked mucosal hyperplasia was scored as 3; and all of the earlier described plus crypt abscesses or ulceration were scored as 4, with a histologic score from 0 to 15.For treatment studies, JAM-A+/+ mice undergoing DSS treatment were injected intraperitoneally at day −3, 0, 3, and 6 with a dose of 100 μg/mouse of monoclonal antibody BV11 in a pyrogen-free saline. In some experiments, mice underwent a combination of ciprofloxacin (50 mg/kg/day) and metronidazole (100 mg/kg/day) for 4 weeks before DSS treatment and during DSS treatment.Endoscopic Damage AssessmentDamage to the colonic mucosa during treatment with DSS was followed up in vivo using the Coloview (Karl Storz, Tuttlingen, Germany) system experimental endoscopy set-up, as previously described.19Nenci A. Becker C. Wullaert A. et al.Epithelial NEMO links innate immunity to chronic intestinal inflammation.Nature. 2007; 446: 557-561Crossref PubMed Scopus (833) Google Scholar This endoscopic procedure was performed under anesthesia. The endoscopic score of colitis severity (murine endoscopic intestinal colitis score: range, 0–15 points) was based on the evaluation of colon translucency (0–3 points), presence of fibrin attached to the bowel wall (0–3 points), granular aspect of the mucosa (0–3 points), morphology of the vascular pattern (0–3 points), and the presence of loose stools (0–3 points).Colon Organ Culture and Enzyme-Linked Immunosorbent AssayColons from all mice were excised, opened, and cut longitudinally into 3 parts. One of all 3 parts was washed in cold phosphate-buffered saline (PBS) supplemented with penicillin, streptomycin, and amphotericin B (Cambrex; BioWhittaker, Walkersville, MD) and incubated in serum-free RPMI 1640 medium with 0.1% fetal bovine serum, penicillin, streptomycin, and amphotericin B at 37°C in 5% CO2. After 24 hours, supernatant fluid was collected, centrifuged, and stored at −20°C. Supernatants were analyzed for tumor necrosis factor α (TNF-α), macrophage inflammatory protein (MIP)-1-α, MIP-2, and keratinocyte chemoattractant (KC) content in duplicate using commercially available enzyme-linked immunosorbent assay kits, as previously reported (R&D Systems, Minneapolis, MN).15Danese S. Fiocchi C. Etiopathogenesis of inflammatory bowel diseases.World J Gastroenterol. 2006; 12: 4807-4812PubMed Google Scholar In some experiments, serum was obtained by centrifugation, stored at −80C, and then cytokine concentration was measured.Western Blot AnalysisWestern blot analysis was performed as previously reported.20Danese S. de la Motte C. Reyes B.M. et al.Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification.J Immunol. 2004; 172: 2011-2015Crossref PubMed Scopus (158) Google ScholarImmunofluorescence StainingCaco-2 cells (40,000 cells/cm2) were seeded on fibronectin-coated (5 μg/mL in PBS; Sigma, St Louis, MO) glass coverslips (13-mm diameter; Carolina Biological Supply Company, Burlington, NY) in 1 mL of complete medium. Cells were fixed on ice with prechilled methanol for 5 minutes and stained with ZO-1 (Zymed) 1:125 or β-catenin (AbCam). Alternatively, for the occludin staining (Zymed), the cells were permeabilized with 0.2% Triton X-100 (2 min on ice), fixed with PFA 3.7% at room temperature (RT) for 30 minutes, and then repermeabilized with 0.05% Triton X-100 (5 min on ice). E-cadherin–stained cells (Zymed) were fixed with prechilled ethanol 95% for 30 minutes at 4°C and then with prechilled acetone for 1 minute at RT. Blocking was performed in 2% bovine serum albumin (Sigma) for 1 hour at RT. After incubation with the primary antibody (2–5 μg/mL in PBS 2% bovine serum albumin) for 1 hour at RT, cells were washed 3 times with PBS 0.2% bovine serum albumin and labeled with the appropriate fluorescently conjugated secondary antibody for 45 minutes at RT.Frozen sections (4-μm) of colonic mucosa from human and murine tissue were fixed in cold acetone (10 minutes at −20°C). The sections then were blocked with PBS containing 2% bovine serum albumin for 60 minutes at RT and incubated with primary antibodies: anti-JAM-A (1:50), E-cadherin (1:50), or ZO-1 (1:125) for 1 hour at RT. Alexa Fluor 488–conjugated goat-antimouse, anti-rat, or anti-rabbit IgG, and/or Alexa Fluor 594–conjugated goat-antimouse or anti-rat IgG antibodies were used as secondary antibodies (1:1000, 30 minutes, RT), followed by incubation with 1 μg/mL Hoechst 33258 (5 minutes, RT), as previously described.18Scaldaferri F. Sans M. Vetrano S. et al.Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease.J Clin Invest. 2007; 117: 1951-1960Crossref PubMed Scopus (105) Google Scholar, 21Danese S. de La Motte C. Sturm A. et al.Platelets trigger a CD40-dependent inflammatory response in the microvasculature of Inflammatory bowel disease patients.Gastroenterology. 2003; 124: 1249-1264Abstract Full Text Full Text PDF PubMed Scopus (178) Google ScholarSections were mounted with FluorSave reagent (Calbiochem, San Diego, CA) and analyzed with a laser scanning confocal microscope (Fluoview FV1000; Olympus, Tokyo, Japan). Images (1024 × 1024 pixels) were acquired with an oil immersion objective (100× 1.4 NA Plan-Apochromat; Olympus). Quantification of JAM-A expression was performed in a blinded manner on stained sections by a quantitative method that assessed the fluorescence intensity in pixels (FV1000 FlowView; Olympus).18Scaldaferri F. Sans M. Vetrano S. et al.Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease.J Clin Invest. 2007; 117: 1951-1960Crossref PubMed Scopus (105) Google Scholar, 22Danese S. Sans M. de la Motte C. et al.Angiogenesis as a novel component of inflammatory bowel disease pathogenesis.Gastroenterology. 2006; 130: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 23Danese S. Sans M. Scaldaferri F. et al.TNF-alpha blockade down-regulates the CD40/CD40L pathway in the mucosal microcirculation: a novel anti-inflammatory mechanism of infliximab in Crohn's disease.J Immunol. 2006; 176: 2617-2624Crossref PubMed Scopus (140) Google Scholar Fluorescence intensity in pixels was quantified in triplicate from each tissue section.Assessment of Apoptosis and Down-Regulation of JAM-A ExpressionThe terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling assay was performed on cryostatic sections using the in situ cell death detection kit (Fluorescein; Roche, Mannheim, Germany) according to the manufacturer's recommendations. For detection of epithelial cells (E-cadherin) and nuclei (Hoechst 33258), immunofluorescence staining were performed in parallel to terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling, as previously reported.24Grabig A. Paclik D. Guzy C. et al.Escherichia coli strain Nissle 1917 ameliorates experimental colitis via Toll-like receptor 2- and Toll-like receptor 4-dependent pathways.Infect Immun. 2006; 74: 4075-4082Crossref PubMed Scopus (157) Google Scholar Apoptosis was quantified as the percentage of apoptotic epithelial cells/field.In vitro apoptosis was studied by flow cytometry and by acridine orange and ethidium bromide staining.25Baskic D. Popovic S. Ristic P. et al.Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide.Cell Biol Int. 2006; 30: 924-932Crossref PubMed Scopus (359) Google ScholarDown-regulation of JAM-A expression was performed as previously described.26Rubinson D.A. Dillon C.P. Kwiatkowski A.V. et al.A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.Nat Genet. 2003; 33: 401-406Crossref PubMed Scopus (1341) Google ScholarEpithelial Transepithelial ResistanceShort hairpin RNA (shRNA)-infected or control Caco-2 cells (4 × 105) (see earlier) were seeded in the upper chamber of a transwell filter (pore diameter, 3 μm; Costar, Milan, Italy) for 10 days. The transepithelial resistance (TER) was measured every other day until day 7 and then daily until day 10 with a Voltmeter (Millicels; Millipore, Bedford, MA), in the presence or absence of 50 nmol/L of the caspase inhibitor Z-VAD (R&D Systems).Statistical AnalysisData were analyzed by Graphpad software (San Diego, CA) and expressed as mean ± SEM. The Kruskal–Wallis test was used for global comparisons within multiple groups, and the Mann–Whitney U test was used for comparison between 2 groups. Statistical significance was set at a P value of less than .05.ResultsEvidence for Down-Regulation of JAM-A in the IBD EpitheliumTo investigate the expression levels of JAM-A in normal and IBD-involved intestine, we performed confocal fluorescence microscopy of 11 histologically control and 13 active CD- and 15 UC-involved mucosa derived from surgical specimens. Normal colonic mucosa showed strong epithelial and vascular immunostaining of JAM-A. The specificity of JAM-A at the epithelial and endothelial junction sites was confirmed by co-staining with the tight junctions marker ZO-1 (Figure 1).In contrast to control intestine (1755 ± 66 fluorescence intensity of pixels), in the mucosa of actively inflamed CD (728 ± 70 fluorescence intensity of pixels) or UC (650 ± 65 fluorescence intensity of pixels) patients, epithelial JAM-A was down-regulated significantly (both P < .01) without any apparent difference between the 2 forms of IBD (Figure 2A), whereas its endothelial expression was similar between controls and IBD patients (not shown). When normal mucosal tissues were compared with uninvolved CD and UC mucosa (1645 ± 70 and 1745 ± 85 fluorescence intensity of pixels, respectively), no significant differences were observed in JAM-A expression (Figure 2A). When assessed by Western blot analysis, JAM-A levels in inflamed mucosa also were significantly lower (P < .05) in mucosal extracts from CD- (0.17 ± 0.01) and UC- (0.28 ± 0.02) involved tissues compared with control tissue (0.67 ± 0.02) (Figure 2B).Figure 2JAM-A expression in IBD mucosa. (A) Fluorescence micrographs of colonic mucosa in (a) normal and actively inflamed (b) UC and (c) CD, and (d) inactive IBD mucosa, and (f–h) their relative H&E stainings. The staining represents the yellow merge of the staining for JAM-A (red), ZO-1 (green), and the nuclei (blue). The panels are representative of 11 controls, 15 UC, and 13 CD samples (magnification, 10×). (B) Western blot for JAM-A in mucosal extracts derived from normal and UC and CD subjects. The blot is representative of 6 controls, 8 UC, and 7 CD samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next investigated JAM-A expression in murine intestine using DSS to induce colitis, a widely used model of experimental IBD. As assessed by confocal microscopy, the murine JAM-A expression pattern was essentially similar to that of human specimens. JAM-A was expressed mainly by epithelial and endothelial cells, as determined by its colocalization with E-cadherin (not shown). However, when compared with the normal epithelium where JAM-A was expressed abundantly, there was a dramatic down-regulation in DSS colitis (Figure 3). No differences were observed in JAM-A expression levels between the control and colitic microvasculature of the murine intestine (not shown).Figure 3JAM-A expression in control and colitic colons in mice. Fluorescence micrographs of murine colonic mucosa in normal and colitic mucosa. Fluorescence micrographs of colons in normal and colitic epithelium, stained for (A and D) JAM-A (red), (B and E) E-cadherin (green), (C and F) nuclei (blue) and merge (yellow). The panels are representative of 7 controls and 8 colitic mice (magnification, 10×).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of Genetic Deletion of JAM-A on Experimental ColitisBecause JAM-A is expressed by both intestinal endothelial and epithelial cells, we investigated the effect of JAM-A deletion in mice administered DSS. We used both mice with a selective endothelial JAM-A deletion (mice Tie-2 Cre JAM-A-/-) and total JAM-A knockout (KO). Of note, Tie-2 also is active in the hematopoietic lineage, leading to Cre expression and deletion of loxP flanked genes in this compartment.27Gitler A.D. Kong Y. Choi J.K. et al.Tie2-Cre-induced inactivation of a conditional mutant Nf1 allele in mouse results in a myeloproliferative disorder that models juvenile myelomonocytic leukemia.Pediatr Res. 2004; 55: 581-584Crossref PubMed Scopus (34) Google Scholar Compared with wild-type mice, Tie-2 Cre JAM-A-/- showed no changes in their susceptibility to DSS-induced colitis over a period of up to 10 days (Figure 4A). On the contrary, mice with a total KO of JAM-A showed a dramatically increased susceptibility (P < .05) to DSS-induced colitis as early as at day 4 compared with the wild-type (wt) mice, as shown by a weight loss and colitic disease activity index (Figure 4A and B). Strikingly, 8 days after 2% DSS treatment, 100% of JAM-A total KO mice died compared with 0% of wt mice (Figure 4C). In addition, mice with a total KO of JAM-A displayed significantly (P < .05) more severe endoscopic damage and a very high histologic inflammation score (Figure 5). No differences in endoscopic and histologic inflammation scores were observed between control wt and JAM-A-/- mice before DSS treatment (Figure 5). Wild-type mice administered DSS and treated with the BV11 monoclonal antibody to block JAM-A displayed a similar phenotype to JAM-A-/- mice, as assessed by weight loss (supplementary Figure 1A; see supplementary material online at www.gastrojournal.org), disease activity index, and histologic scores (not shown).Figure 4(A) Susceptibility to DSS-induced colitis in Tie-2 Cre JAM-A-/- and JAM-A-/- mice. Wt (●, n = 11), Tie-2 Cre JAM-A-/- (▲, n = 12), and JAM-A-/- (■, n = 14) animals underwent DSS treatment for 7 days. Weight was monitored daily. After 7 days, mice were killed and colons were assessed for histologic colitis. *P < .05 for wild-type vs JAM-A-/- mice. (B and C) Susceptibility to DSS-induced colitis in JAM-A-/- mice. Wt (n = 11) or JAM-A-/- (n = 14) animals underwent DSS treatment for 10 days. (B) Clinical disease activity index was calculated daily, and (C) survival was monitored. *P < .05 for wt vs JAM-A-/- mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Endoscopic and histologic colitis in JAM-A-/- mice. Wt (n = 11) or JAM-A-/- (n = 14) animals underwent DSS treatment for 7 days. An endoscopy was performed at the beginning and after 7 days of DSS in (A and C) wt and (B and D) JAM-A-/- and an endoscopic score was calculated. After endoscopy, wt and JAM-A-/- mice were sacrificed. An histologic colitis score was calculated at the beginning and after 7 days of DSS in wt (E and G) and JAM-A-/- (F and H). *P < .05 for wt vs JAM-A-/- mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To rule out the possible involvement of the intestinal flora in the increased susceptibility to intestinal inflammation, experiments were run in parallel in JAM-A-/- and wt mice administered antibiotics. No differences were found in t
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