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• W2024942721 abstract "The E-cadherin receptor CD103 (αEβ7-integrin) is expressed on specific populations of pulmonary dendritic cells (DC) and T cells. However, CD103 function in the lung is not well understood. Matrilysin (MMP-7) expression is increased in lung injury and cleaves E-cadherin from injured lung epithelium. Thus, to assess matrilysin effects on CD103-E-cadherin interactions in lung injury, wild-type, CD103−/−, and Mmp7−/− mice, in which E-cadherin isn’t cleaved in the lung, were treated with bleomycin or bleomycin with nFMLP to reverse the defect in acute neutrophil influx seen in Mmp7−/− mice. Pulmonary CD103+ DC were significantly increased in injured wild-type compared with Mmp7−/− mice, and CD103+ leukocytes showed significantly enhanced interaction with E-cadherin on injured wild-type epithelium than with Mmp7−/− epithelium in vitro and in vivo. Bleomycin-treated CD103−/− mice had persistent neutrophilic inflammation, increased fibrosis, and increased mortality compared with wild-type mice, a phenotype that was partially recapitulated in bleomycin/nFMLP-treated Mmp7−/− mice. Soluble E-cadherin increased IL-12 and IL-10 and reduced IL-6 mRNA expression in wild-type bone marrow-derived DC but not in CD103−/− bone marrow-derived DC. Similar mRNA patterns were seen in lungs of bleomycin-injured wild-type, but not CD103−/− or Mmp7−/−, mice. In conclusion, matrilysin regulates pulmonary localization of DC that express CD103, and E-cadherin cleavage may activate CD103+ DC to limit inflammation and inhibit fibrosis. The E-cadherin receptor CD103 (αEβ7-integrin) is expressed on specific populations of pulmonary dendritic cells (DC) and T cells. However, CD103 function in the lung is not well understood. Matrilysin (MMP-7) expression is increased in lung injury and cleaves E-cadherin from injured lung epithelium. Thus, to assess matrilysin effects on CD103-E-cadherin interactions in lung injury, wild-type, CD103−/−, and Mmp7−/− mice, in which E-cadherin isn’t cleaved in the lung, were treated with bleomycin or bleomycin with nFMLP to reverse the defect in acute neutrophil influx seen in Mmp7−/− mice. Pulmonary CD103+ DC were significantly increased in injured wild-type compared with Mmp7−/− mice, and CD103+ leukocytes showed significantly enhanced interaction with E-cadherin on injured wild-type epithelium than with Mmp7−/− epithelium in vitro and in vivo. Bleomycin-treated CD103−/− mice had persistent neutrophilic inflammation, increased fibrosis, and increased mortality compared with wild-type mice, a phenotype that was partially recapitulated in bleomycin/nFMLP-treated Mmp7−/− mice. Soluble E-cadherin increased IL-12 and IL-10 and reduced IL-6 mRNA expression in wild-type bone marrow-derived DC but not in CD103−/− bone marrow-derived DC. Similar mRNA patterns were seen in lungs of bleomycin-injured wild-type, but not CD103−/− or Mmp7−/−, mice. In conclusion, matrilysin regulates pulmonary localization of DC that express CD103, and E-cadherin cleavage may activate CD103+ DC to limit inflammation and inhibit fibrosis. Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute epithelial and endothelial damage, leakage of proteinaceous edema fluid into the alveolar space and interstitium, and a leukocytic cellular infiltrate, with polymorphonuclear neutrophils being the key inflammatory cell population in both humans and in experimental animals.1Ware LB Matthay MA The acute respiratory distress syndrome.N Engl J Med. 2000; 342: 1334-1349Crossref PubMed Scopus (4430) Google Scholar Unfavorable outcomes in patients with ALI/acute respiratory distress syndrome are associated with an exaggerated pulmonary inflammatory response that persists unabated over time.2Goodman RB Strieter RM Martin DP Steinberg KP Milberg JA Maunder RJ Kunkel SL Walz A Hudson LD Martin TR Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome.Am J Respir Crit Care Med. 1996; 154: 602-611Crossref PubMed Scopus (465) Google Scholar, 3Meduri GU Headley S Kohler G Stentz F Tolley E Umberger R Leeper K Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS: plasma IL-1β and IL-6 levels are consistent and efficient predictors of outcome over time.Chest. 1995; 107: 1062-1073Crossref PubMed Scopus (628) Google Scholar Failure to resolve acute inflammation also contributes to chronic lung injury and pulmonary fibrosis, and the presence of extensive fibrosis may be an independent risk factor that correlates with poor outcome.4Ichikado K Suga M Muranaka H Gushima Y Miyakawa H Tsubamoto M Johkoh T Hirata N Yoshinaga T Kinoshita Y Yamashita Y Sasaki Y Prediction of prognosis for acute respiratory distress syndrome with thin-section CT: validation in 44 cases.Radiology. 2006; 238: 321-329Crossref PubMed Scopus (139) Google Scholar Impaired epithelial repair contributes to fibrosis in the lung, liver, kidney, and other tissues,5Selman M Pardo A Idiopathic pulmonary fibrosis: an epithelial/fibroblastic cross-talk disorder.Resp Res. 2002; 3: 1-8Crossref PubMed Scopus (91) Google Scholar, 6Crystal RG Bitterman PB Mossman B Schwartz MI Sheppard D Almasy L Chapman HA Friedman SL King TEJ Leinwald LA Loiotta L Martin GR Schwartz DA Schultz GS Wagner CR Musson RA Future research direction in idiopathic pulmonary fibrosis: summary of a National Heart, Lung, and Blood Institute working group.Am J Respir Crit Care Med. 2002; 166: 236-246Crossref PubMed Scopus (162) Google Scholar and epithelial cell interactions with inflammatory and mesenchymal cells are central to both physiological lung repair and pathological lung remodeling.Important among the pulmonary responses to injury is the increased expression and activation of enzymes in the matrix metalloproteinase (MMP) family.7Greenlee KJ Werb Z Kheradmand F Matrix metalloproteinases in lung: multiple, multifarious, and multifaceted.Physiol Rev. 2007; 87: 69-98Crossref PubMed Scopus (341) Google Scholar MMPs are zinc-binding enzymes with activity against a wide range of extracellular proteins,8Brinckerhoff CE Matrisian LM Matrix metalloproteinases: a tail of a frog that became a prince.Nat Rev Mol Cell Biol. 2002; 3: 207-214Crossref PubMed Scopus (959) Google Scholar and MMP expression is typically limited to tissue remodeling associated with development, involution, inflammation, tumor growth, and repair. Our laboratory found that matrilysin (MMP-7) is strongly induced in injured alveolar epithelium in emphysema, desquamative interstitial pneumonitis, cystic fibrosis, and acute respiratory distress syndrome.9Dunsmore SE Saarialho-Kere UK Roby JD Wilson CL Matrisian LM Welgus HG Parks WC Matrilysin expression and function in airway epithelium.J Clin Invest. 1998; 102: 1321-1331Crossref PubMed Scopus (231) Google Scholar, 10McGuire JK Li QL Parks WC Matrilysin mediates E-cadherin ectodomain shedding in injured lung epithelium.Am J Pathol. 2003; 162: 1843-1861Abstract Full Text Full Text PDF Scopus (257) Google Scholar In bleomycin-induced lung injury in mice, matrilysin expression is increased in alveolar epithelium early after injury and regulates acute neutrophil influx by controlling KC chemokine release into the alveolar compartment during the first 5 days following injury.11Li QL Park PY Wilson CL Parks WC Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury.Cell. 2002; 111: 635-646Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar Beyond the acute phase of injury, matrilysin expression increases as neutrophilic inflammation subsides and fibrosis ensues,11Li QL Park PY Wilson CL Parks WC Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury.Cell. 2002; 111: 635-646Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar and thus, matrilysin has been implicated in the progression of pulmonary fibrosis.12Zuo F Kaminski N Eugui E Allard J Yakhini Z Ben-Dor A Lollini L Morris D Kim Y DeLustro B Sheppard D Pardo A Selman M Heller RA Gene expression analysis reveal matrilysin as a key regulator of pulmonary fibrosis in mice and humans.Proc Natl Acad Sci USA. 2002; 99: 6292-6297Crossref PubMed Scopus (517) Google Scholar However, when acute neutrophil influx is restored in bleomycin-treated matrilysin-null (Mmp7−/−) mice with the neutrophil chemotactic peptide nFMLP, mortality is higher in Mmp7−/− mice than in wild-type mice.11Li QL Park PY Wilson CL Parks WC Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury.Cell. 2002; 111: 635-646Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar Thus, observations of increased fibrosis in bleomycin-treated Mmp7−/− mice likely reflect the early acute injury phenotype, and in chronic lung injury, matrilysin activity may regulate physiological functions that promote repair.E-cadherin regulates cell-cell adhesion in most epithelia and maintains epithelial integrity, restricts migration and proliferation, and promotes differentiation.13Yap AS Brieher WM Gumbiner BM Molecular and functional analysis of cadherin-based adherens junctions.Annu Rev Cell Dev Biol. 1997; 13: 119-146Crossref PubMed Scopus (685) Google Scholar The proteolytic cleavage of membrane proteins from the cell surface has been described as “ectodomain shedding,”14Arribas J Borroto A Protein ectodomain shedding.Chem Rev. 2002; 102: 4627-4638Crossref PubMed Scopus (205) Google Scholar, 15Dello Sbarba P Rovida E Transmodulation of cell surface regulatory molecules via ectodomain shedding.Biol Chem. 2002; 383: 69-83PubMed Google Scholar, 16Garton KJ Gough PJ Raines EW Emerging roles for ectodomain shedding in the regulation of inflammatory responses.J Leukoc Biol. 2006; 79: 1105-1116Crossref PubMed Scopus (187) Google Scholar, 17Reiss K Saftig P The “a disintegrin and metalloprotease” (ADAM) family of sheddases: physiological and cellular functions.Semin Cell Dev Biol. 2009; 20: 126-137Crossref PubMed Scopus (323) Google Scholar and we described a physiological role for matrilysin-dependent shedding of the E-cadherin ectodomain in airway mucosal repair.10McGuire JK Li QL Parks WC Matrilysin mediates E-cadherin ectodomain shedding in injured lung epithelium.Am J Pathol. 2003; 162: 1843-1861Abstract Full Text Full Text PDF Scopus (257) Google Scholar We also found that matrilysin cleaves E-cadherin from alveolar epithelium during the progression of bleomycin-induced pulmonary fibrosis, and Mmp7−/− mice do not shed E-cadherin in the injured lung. A few in vitro studies have evaluated the function of E-cadherin shedding in cancer cells, suggesting potential roles in regulating cancer cell migration or gene expression.18Noe V Fingleton B Jacobs K Crawford HC Vermeulen S Steelant W Bruyneel E Matrisian LM Mareel M Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1.J Cell Sci. 2001; 114: 111-118Crossref PubMed Google Scholar, 19Nawrocki-Raby B Gilles C Pollette M Bruyneel E Laronze J-Y Bonnet N Foidart J-M Mareel M Birembaut P Up-regulation of MMPs by soluble E-cadherin in human lung tumor cells.Int J Cancer. 2003; 105: 790-795Crossref PubMed Scopus (103) Google Scholar However, to our knowledge, in vivo functions for E-cadherin shedding in chronic lung injury and fibrosis have not been previously assessed.The leukocyte-specific αEβ7-integrin (CD103) is expressed on nearly all intraepithelial lymphocytes and on specific populations of dendritic cells (DC), and E-cadherin is the only known CD103 ligand.20Kilshaw PJ αEβ7.Mol Pathol. 1999; 52: 203-207Crossref PubMed Scopus (52) Google Scholar, 21Taraszka KS Higgins JM Tan K Mandelbrot DA Wang JH Brenner MB Molecular basis for leukocyte integrin αEβ7 adhesion to epithelial (E)-cadherin.J Exp Med. 2000; 191: 1555-1567Crossref PubMed Scopus (57) Google Scholar Transforming growth factor-β1 (TGF-β1) induces CD103 expression, and increased TGF-β1 in injured tissues may up-regulate CD103 on infiltrating leukocytes.22El-Asady R Yuan R Liu K Wang D Gress RE Lucas PJ Drachenberg CB Hadley G TGF-β-dependent CD103 expression by CD8+ T cells promotes selective destruction of the host intestinal epithelium during graft-versus-host disease.J Exp Med. 2005; 201: 1647-1657Crossref PubMed Scopus (201) Google Scholar, 23Ling KL Dulphy N Bahl P Salio M Maskell K Piris J Warren BF George BD Mortensen NJ Cerundolo V Modulation of CD103 expression on human colon carcinoma-specific CTL.J Immunol. 2007; 178: 2908-2915PubMed Google Scholar, 24Wang D Yuan R Feng Y El-Asady R Farber DL Gress RE Lucas PJ Hadley GA Regulation of CD103 expression by CD8+ T cells responding to renal allografts.J Immunol. 2004; 172: 214-221PubMed Google Scholar Via interaction with E-cadherin, CD103 has been suggested to be an epithelial recognition molecule that retains CD103+ lymphocytes at epithelial surfaces, targets epithelial tumor cells for destruction by cytolytic T cells, or regulates kidney allograft rejection.23Ling KL Dulphy N Bahl P Salio M Maskell K Piris J Warren BF George BD Mortensen NJ Cerundolo V Modulation of CD103 expression on human colon carcinoma-specific CTL.J Immunol. 2007; 178: 2908-2915PubMed Google Scholar, 25Schon MP Arya A Murphy EA Adams CM Strauch UG Agace WW Marsal J Donohue JP Her H Beier DR Olson S Lefrancois L Brenner MB Grusby MJ Parker CM Mucosal T lymphocyte numbers are selectively reduced in integrin αE (CD103)-deficient mice.J Immunol. 1999; 162: 6641-6649PubMed Google Scholar, 26Le Floc'h A Jalil A Vergnon I Le Maux Chansac B Lazar V Bismuth G Chouaib S Mami-Chouaib F αEβ7 integrin interaction with E-cadherin promotes antitumor CTL activity by triggering lytic granule polarization and exocytosis.J Exp Med. 2007; 204: 559-570Crossref PubMed Scopus (185) Google Scholar, 27Smyth LJ Kirby JA Cunningham AC Role of the mucosal integrin αE(CD103)β7 in tissue-restricted cytotoxicity.Clin Exp Immunol. 2007; 149: 162-170Crossref PubMed Scopus (27) Google Scholar, 28Hadley G Role of integrin CD103 in promoting destruction of renal allografts by CD8+ T cells.Am J Transplant. 2004; 4: 1026-1032Crossref PubMed Scopus (20) Google Scholar CD103+ pulmonary DC arise from myeloid mononuclear precursors, do not express plasmacytoid DC markers,29Sung SS Fu SM Rose Jr, CE Gaskin F Ju ST Beaty SR A major lung CD103 (αE)-β7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins.J Immunol. 2006; 176: 2161-2172PubMed Google Scholar, 30Jakubzick C Tacke F Ginhoux F Wagers AJ van Rooijen N Mack M Merad M Randolph GJ Blood monocyte subsets differentially give rise to CD103+ and CD103− pulmonary dendritic cell populations.J Immunol. 2008; 180: 3019-3027PubMed Google Scholar and appear to have distinct cytokine and antigen presentation capabilities compared with CD103− myeloid DC populations.31Beaty SR Rose Jr, CE Sung SS Diverse and potent chemokine production by lung CD11bhigh dendritic cells in homeostasis and in allergic lung inflammation.J Immunol. 2007; 178: 1882-1895PubMed Google Scholar, 32del Rio ML Rodriguez-Barbosa JI Kremmer E Forster R CD103− and CD103+ bronchial lymph node dendritic cells are specialized in presenting and cross-presenting innocuous antigen to CD4+ and CD8+ T cells.J Immunol. 2007; 178: 6861-6866PubMed Google Scholar However, the function of CD103 in lung injury has not been defined. Therefore, we explored the possibility that E-cadherin shedding could be a mechanism controlling interactions between leukocytes that express CD103 and epithelial cells that express its ligand E-cadherin in bleomycin-induced lung injury in mice.Materials and MethodsAnimals and Bleomycin Lung Injury ModelMice carrying a targeted deletion of the matrilysin gene on the C57BL/6 background33Wilson CL Heppner KJ Labosky PA Hogan BLM Matrisian LM Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin.Proc Natl Acad Sci USA. 1997; 94: 1402-1407Crossref PubMed Scopus (545) Google Scholar are maintained in our laboratory and are designated Mmp7−/− mice. Mice carrying a targeted deletion of the αE-integrin subunit gene (Itgae) on the C57BL/6 background34Yuan R El-Asady R Liu K Wang D Drachenberg CB Hadley GA Critical role for CD103+CD8+ effectors in promoting tubular injury following allogeneic renal transplantation.J Immunol. 2005; 175: 2868-2879PubMed Google Scholar were proved by G. Hadley (Department of Surgery, Ohio State University, Columbus, OH) and are designated CD103−/− mice. Because αE-integrin subunits partner only with β7-integrin subunits, deficiency of the αE-integrin subunit only affects the αEβ7-integrin, and expression or function of other integrins is not affected.35Kilshaw PJ Murant SJ Expression and regulation of β7(β p) integrins on mouse lymphocytes: relevance to the mucosal immune system.Eur J Immunol. 1991; 21: 2591-2597Crossref PubMed Scopus (204) Google Scholar Homozygous CD103 deficiency does not affect fecundity, morphogenesis, growth, or postnatal survival in mice raised in a specific pathogen free animal facility.25Schon MP Arya A Murphy EA Adams CM Strauch UG Agace WW Marsal J Donohue JP Her H Beier DR Olson S Lefrancois L Brenner MB Grusby MJ Parker CM Mucosal T lymphocyte numbers are selectively reduced in integrin αE (CD103)-deficient mice.J Immunol. 1999; 162: 6641-6649PubMed Google Scholar Control mice were congenic C57BL/6 wild-type mice. For bleomycin instillation, age- and sex-matched wild-type and transgenic mice (8 to 10 weeks of age) were anesthetized with i.p. injection of 2.5% Avertin in 0.9% normal saline, net dose 500 mg/kg body weight (Sigma-Aldrich, St. Louis, MO), and tracheas were cannulated via endotracheal intubation by passing a blunt tip 22-gauge angiocatheter through the vocal cords using direct visualization. Bleomycin was diluted in normal saline to provide a dose of 1.25 units/kg body weight in 50-μl volume, which was administered via a bolus into the hub of the angiocatheter and aspirated into the lungs of the spontaneously breathing, anesthetized mice. Animals were allowed to recover from anesthesia and take food and water ad libitum. At specified time points after injury, animals were euthanized by carbon dioxide asphyxiation, and thoracotomy was performed. Tracheas were cannulated with a 22-gauge 1-in angiocatheter (BD Biosciences, San Diego, CA), and bronchoalveolar lavage (BAL) was collected in 2 × 0.8-ml aliquots of sterile 0.9% saline solution. Lungs were removed and either homogenized in RNeasy RLT buffer (Qiagen, Germantown, MD) for RNA isolation or 0.5 M acetic acid for collagen quantification, snap-frozen in optimal cutting temperature tissue freezing medium for immunohistochemistry, or prepared for flow cytometry analysis as described below. All experiments were performed with the approval of the University of Washington School of Medicine Institutional Animal Use and Care Committee.AntibodiesMonoclonal antibodies against mouse antigens CD16/CD32 (Fc-block, clone 2.4G2), CD45 (30-F11), and CD103 (M290) were obtained from BD Biosciences and against CD103 (2E7), CD11c (N418), Langerin (eBioL31), and CD3e (500A2) were obtained from eBioscience (San Diego, CA). Anti-mouse E-cadherin (ECCD-2) was obtained from Invitrogen (Carlsbad, CA). Anti-mouse F4/80 (MCA497BB) was obtained from AbD Serotec (Raleigh, NC). Fluorescent-conjugated anti-Armenian hamster secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA), and anti-rat fluorescent-conjugated antibodies were from Invitrogen (Molecular Probes).ImmunohistochemistryFive-micrometer sections were cut from blocks of frozen lung tissues, air-dried for 5 minutes, fixed in 75% acetone/25% ethanol, washed in PBS, and blocked with TBST (25 mmol/L Tris-HCL (pH 7.5), 150 mmol/L NaCl, and 0.1% Tween 20) containing 1% BSA and 2% goat serum. Fixed tissues or airway epithelial cell cultures were incubated with primary antibodies diluted in 1% BSA/PBS for 60 minutes at 37°C, washed with PBS, and incubated with appropriate fluorescent-conjugated secondary antibodies diluted in 1% BSA/PBS at the manufacturer’s recommended concentrations for 60 minutes at 37°C, followed by washing and mounting with ProLong Gold Antifade (Invitrogen). Immunofluorescence images of tissues and cells were captured with an Olympus DP70 digital camera system (Olympus America, Center Valley, PA).Cell CultureAll cell culture media and supplements were obtained from Mediatech (Herndon, VA) unless otherwise specified. Air-liquid interface airway epithelial cultures were established from wild-type and matrilysin-null mice and maintained as described previously.36Kassim SY Gharib SA Mecham BH Birkland TP Parks WC McGuire JK Individual matrix metalloproteinases control distinct transcriptional responses in airway epithelial cells infected with Pseudomonas aeruginosa.Infect Immun. 2007; 75: 5640-5650Crossref PubMed Scopus (61) Google Scholar MTC-1 cells, a mouse T-hybridoma line, were a gift from P. Kilshaw (Babraham Institute, Cambridge, UK) and were maintained in RMPI 1640 supplemented with 10% FCS and 5 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN) with CD103 expression verified by flow cytometry. Wounds were made in confluent air-liquid interface cultures grown in 6.5-mm Transwells (Corning Life Sciences, Lowell, MA) by gently scratching with a P1000 pipette tip, rinsing with sterile PBS and refeeding with fresh medium. A total of 103 MTC-1 cells were suspended in PBS and added to the apical chamber of wounded cultures and allowed to incubate overnight under standard culture conditions. The next day, cultures were vigorously washed three times with sterile PBS, fixed for immunostaining in 100% methanol, followed by washing with PBS, blocking with PBS supplemented with 1% BSA, and staining for E-cadherin and CD103. After immunostaining, membranes were removed from Transwells and mounted onto glass slides with Prolong Gold Antifade, and uniform 250-μm-wide images of the wound edges were digitally captured. The adherent MTC-1 cells per 250-μm wound edge visualized in each image were counted, and the number adherent cells per millimeter of wound was calculated.Bone marrow-derived DC (BMDC) were cultured from femurs of wild-type and CD103−/− mice. Briefly, femurs were removed and cleaned of all excess tissue, ends were cut, and bone marrow was obtained by sterilely flushing marrow cavity with RPMI 1640 and filtering marrow through 70-μm mesh filters to remove debris. Cells were counted and resuspended at 2 × 106 cells/ml in RPMI 1640 supplemented with sodium pyruvate, nonessential amino acids, penicillin/streptomycin, l-glutamine, 10% FBS (HyClone, Logan, UT), and 20 ng/ml GM-CSF (RDI, Concord MA) and seeded in 6-well cell culture plates for 6 days before maturation with 5 ng/ml lipopolysaccharide (LPS from E. coli O111:B4; List Biological Labs, Campbell, CA) for 24 hours. Two days before LPS addition, 5 ng/ml TGF-β1 was added to induce expression of CD103 on BMDC. Flow cytometry confirmed that CD103 was highly expressed on 30 to 35% of CD11c+ BMDC compared with 5 to 6% on BMDC that did not receive TGF-β1. Soluble recombinant mouse E-cadherin/human Fc chimeric protein (R&D Biosystems) was added to BMDC cultures at a final concentration of 100 ng/ml at the time of LPS addition. The chimeric protein contains human IgG1 residues 100–330, and because the mouse Fc receptor binding site on human IgG is at residues 341–439, the Fc portion does not interact with BMDC or other mouse cells that express Fc receptors.37Klein M Neauport-Sautes C Ellerson JR Fridman WH Binding site of human IgG subclasses and their domains for Fc receptors of activated murine T cells.J Immunol. 1977; 119: 1077-1083PubMed Google Scholar, 38Ratcliffe A Stanworth DR The localization of the binding site(s) on human IgG1 for the Fc receptors on homologous monocytes and heterologous mouse macrophages.Immunology. 1983; 50: 93-100PubMed Google ScholarTotal Lung Collagen AssayTotal lung collagen was determined using the picrosirius red Sircol Assay (Biocolor, Carrickfergus, County Antrim, UK) per the manufacturer’s protocol. Briefly, at specified time points after bleomycin administration, mice were euthanized, and the left lung was removed and homogenized in 0.5 M acetic acid solution. A total of 200 μl of the acid homogenate was digested by adding 1 ml of pepsin solution (2 mg/ml in 0.5 M acetic acid) and incubating overnight at 4°C with continuous shaking. After digestion, samples were centrifuged, and 100 μl of the supernatant containing soluble collagen was incubated with 1 ml of Sircol dye reagent for 30 minutes at room temperature. Samples were centrifuged, the supernatant was discarded, and the precipitated pellet was resuspended in 1 ml of Sircol alkali reagent. Collagen concentration was then determined by spectrophotometric absorbance at 540 nm as compared with a standard curve.Western BlottingFor detection of E-cadherin in culture medium from wounded ALI epithelial cell cultures, equal volumes (1 ml) of fresh medium was added to each well at the time of wounding. At 24 hours after wounding, condition media samples were collected from each well, and a 200-μl aliquot of each sample was concentrated 10-fold with Centricon spin concentrator columns (Millipore, Billerica, MA). The entire volume of concentrated sample (∼20 μl) was resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were blocked overnight with 5% dry milk in TBST, probed with ECCD-2 rat anti-mouse E-cadherin at 1/1000 dilution, and developed with Pierce Super Signal West Pico Chemiluminescence substrate (Thermo Scientific, Rockford, IL). Chemiluminescence images were captured with a digital gel documentation system (UVP Bioimaging Systems, Upland, CA).Flow CytometryMouse lungs were minced into 1-mm pieces with scissors and digested with 2 mg/ml bacterial collagenase (Roche, Indianapolis, IN) and 0.5 mg/ml DNase (Sigma-Aldrich) for 1 hour at 37°C on a rocking platform. Digested lung was filtered through a 40-μm nylon cell strainer to form a single-cell suspension. Lung digest or BAL cells were washed with flow cytometry buffer (0.5% BSA in PBS), counted, and blocked with rat IgG2b anti-mouse CD16/CD32 mAb (BD Biosciences) 1 μg per 106 cells. Cells were stained with conjugated primary antibodies for 60 minutes on ice, washed with flow cytometry buffer twice, and analyzed using the Beckman Coulter FC500 Flow Cytometer and CXP Analysis software.Quantitative PCRTotal RNA was isolated with Qiagen RNeasy kits according to manufacturers protocols. Primers and TaqMan probes (FAM dye-labeled) for a1(I) procollagen, interleukin-12 (IL-12) (p40 subunit), interferon γ, IL-10, TGF-β1, connective tissue growth factor, and glyceraldehyde-3-phosphodehydrogenase were added to cDNA synthesized from 5 μg of total RNA with a High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA), and product amplification was measured with an ABI HT7900 Fast Real-Time PCR System. The threshold cycle (Ct) was obtained from duplicate aliquots of each sample and averaged. The ΔCt was the difference between the average Ct for the specific cDNA and the average Ct for glyceraldehyde-3-phosphodehydrogenase. The ΔΔCt, or the difference between ΔCt of the experimental and that of control condition, was calculated as the average ΔCt at a given time point minus the average ΔCt of day 0 samples. The data are expressed as fold change relative to the control condition and was calculated as 2−ΔΔCt.ResultsMatrilysin Regulates Pulmonary Influx of Leukocytes Expressing CD103Matrilysin cleaves the CD103-ligand E-cadherin; therefore, we assessed CD103-expressing leukocytes in bleomycin/nFMLP-treated wild-type compared with Mmp7−/− mice, in which E-cadherin is not cleaved or “shed” from the cell surface.10McGuire JK Li QL Parks WC Matrilysin mediates E-cadherin ectodomain shedding in injured lung epithelium.Am J Pathol. 2003; 162: 1843-1861Abstract Full Text Full Text PDF Scopus (257) Google Scholar To evaluate matrilysin function independent of its role in regulating the early acute neutrophil influx, wild-type mice and Mmp7−/− mice were cotreated with bleomycin and 1 μmol/L nFMLP, which reverses the defect in early neutrophil influx.11Li QL Park PY Wilson CL Parks WC Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury.Cell. 2002; 111: 635-646Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar A low dose of bleomycin was used (1.25 units/kg body weight) that resulted in minimal mortality in mice of either genotype. In normal lungs of both genotypes, CD103 staining was observed on rare cells associated with large airway epithelium, consistent with localization to intraepithelial lymphocytes or resident subepithelial dendritic cells (Figure 1). At 7 days after bleomycin, CD103+ cells were prominently seen in the subepithelial compartment around airways and alveolar spaces of wild-type lungs and in areas of nascent fibrosis, whereas in Mmp7−/− lungs, CD103+ cells were fewer in number and were mainly localized around larger blood vessels. By 14 days after bleomycin, CD103+ cells were seen in areas of fibrosis in both genotypes; however, more CD103+ cells were observed in lungs from wild-type mice than in Mmp7−/− lungs. There were no differences in numbers of CD103+ cells in lungs of uninjured mice or in numbers or localization of intestinal CD103+ IEL cells (data not shown), indicating that there was no generalized defect in CD103+ expression or ability of CD103+ leukocytes to migrate into tissues in Mmp7−/− mice.Flow cytometry was used to quantify differences in CD103+ cells in single-cell suspensions of collagenase/DNase-digested" @default.
• W2024942721 created "2016-06-24" @default.
• W2024942721 creator A5050194774 @default.
• W2024942721 creator A5058402241 @default.
• W2024942721 creator A5068050504 @default.
• W2024942721 date "2009-12-01" @default.
• W2024942721 modified "2023-10-11" @default.
• W2024942721 title "Matrilysin (Matrix Metalloproteinase-7) Regulates Anti-Inflammatory and Antifibrotic Pulmonary Dendritic Cells That Express CD103 (αEβ7-Integrin)" @default.
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