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• W2003443332 abstract "Acidic mammalian chitinase (AMCase) is expressed in an exaggerated fashion in epithelial cells at sites of pulmonary T helper cell type 2 inflammation and plays important roles in the pathogenesis of anti-parasite and asthma-like responses. However, the mechanisms that control epithelial cell AMCase secretion and its effector responses have not been adequately defined. To address these issues, we used in vivo and in vitro experimental systems to define the pathways of epithelial AMCase secretion and its epithelial regulatory effects. Here we demonstrate that, in murine T helper cell type 2 modeling systems, AMCase colocalizes with the epidermal growth factor receptor (EGFR) and ADAM17 (a membrane disintegrin and metallopeptidase 17) in lung epithelial cells. In vitro cotransfection experiments in A549 cells demonstrated that AMCase and EGFR physically interact with each other. Cotransfection of AMCase and EGFR also increased, whereas EGFR inhibition decreased AMCase secretion. Interestingly, AMCase secretion was not significantly altered by treatment with EGF but was significantly decreased when the upstream EGFR transactivator ADAM17 was inhibited. AMCase secretion was also decreased when the EGFR-downstream Ras was blocked. Transfected and recombinant AMCase induced epithelial cell production of CCL2, CCL17, and CXCL8. These studies demonstrate that lung epithelial cells secrete AMCase via an EGFR-dependent pathway that is activated by ADAM17 and mediates its effects via Ras. They also demonstrate that the AMCase that is secreted feeds back in an autocrine and/or paracrine fashion to stimulate pulmonary epithelial cell chemokine production. Acidic mammalian chitinase (AMCase) is expressed in an exaggerated fashion in epithelial cells at sites of pulmonary T helper cell type 2 inflammation and plays important roles in the pathogenesis of anti-parasite and asthma-like responses. However, the mechanisms that control epithelial cell AMCase secretion and its effector responses have not been adequately defined. To address these issues, we used in vivo and in vitro experimental systems to define the pathways of epithelial AMCase secretion and its epithelial regulatory effects. Here we demonstrate that, in murine T helper cell type 2 modeling systems, AMCase colocalizes with the epidermal growth factor receptor (EGFR) and ADAM17 (a membrane disintegrin and metallopeptidase 17) in lung epithelial cells. In vitro cotransfection experiments in A549 cells demonstrated that AMCase and EGFR physically interact with each other. Cotransfection of AMCase and EGFR also increased, whereas EGFR inhibition decreased AMCase secretion. Interestingly, AMCase secretion was not significantly altered by treatment with EGF but was significantly decreased when the upstream EGFR transactivator ADAM17 was inhibited. AMCase secretion was also decreased when the EGFR-downstream Ras was blocked. Transfected and recombinant AMCase induced epithelial cell production of CCL2, CCL17, and CXCL8. These studies demonstrate that lung epithelial cells secrete AMCase via an EGFR-dependent pathway that is activated by ADAM17 and mediates its effects via Ras. They also demonstrate that the AMCase that is secreted feeds back in an autocrine and/or paracrine fashion to stimulate pulmonary epithelial cell chemokine production. Allergic asthma is characterized by an exaggerated T helper cell type 2 (Th2) 3The abbreviations used are: Th2T helper cell type 2AMCaseacidic mammalian chitinaseEGFRepidermal growth factor receptorTGF-αtransforming growth factor-αILinterleukinEGFRepidermal growth factor receptorERKextracellular signal-regulated kinasePBSphosphate-buffered salineWTwild typeOVAovalbumin. immune response with eosinophil-rich tissue and bronchoalveolar lavage inflammation, mucus metaplasia, airway remodeling, and airway hyperresponsiveness. Studies from our laboratory and others have demonstrated that the Th2-associated cytokine interleukin-13 (IL-13) is a central regulator of these responses (1Elias J.A. Lee C.G. Zheng T. Shim Y. Zhu Z. Am. J. Respir. Cell Mol. Biol. 2003; 28: 401-404Crossref PubMed Scopus (34) Google Scholar, 2Elias J.A. Lee C.G. Zheng T. Ma B. Homer R.J. Zhu Z. J. Clin. Invest. 2003; 111: 291-297Crossref PubMed Scopus (383) Google Scholar, 3Elias J.A. Zheng T. Lee C.G. Homer R.J. Chen Q. Ma B. Blackburn M. Zhu Z. Chest. 2003; 123: 339S-345SAbstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In accord with its importance, the mechanisms that IL-13 uses to induce these responses have been intensively investigated. These studies demonstrated that the effects of IL-13 are mediated by a variety of downstream mediators, including CC chemokines, TGF-β1, adenosine and adenosine receptors, vascular endothelial growth factor, IL-11, and the 18-glycosyl hydrolase protein, acidic mammalian chitinase (AMCase) (1Elias J.A. Lee C.G. Zheng T. Shim Y. Zhu Z. Am. J. Respir. Cell Mol. Biol. 2003; 28: 401-404Crossref PubMed Scopus (34) Google Scholar, 4Elias J.A. Homer R.J. Hamid Q. Lee C.G. J. Allergy Clin. Immunol. 2005; 116: 497-500Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 5Zhu Z. Lee C.G. Zheng T. Chupp G. Wang J. Homer R.J. Noble P.W. Hamid Q. Elias J.A. Am. J. Respir. Crit. Care Med. 2001; 164: S67-S70Crossref PubMed Scopus (87) Google Scholar, 6Zhu Z. Zheng T. Homer R.J. Kim Y.K. Chen N.Y. Cohn L. Hamid Q. Elias J.A. Science. 2004; 304: 1678-1682Crossref PubMed Scopus (697) Google Scholar). The last is particularly intriguing, because these studies demonstrated that (a) AMCase is induced in epithelial cells and macrophages at sites of Th2 inflammation, (b) IL-13 is necessary and sufficient to stimulate AMCase production, (c) exaggerated AMCase expression can be readily appreciated in biopsies from patients with asthma, and (d) anti-AMCase-based interventions ameliorate Th2 inflammation and physiologic dysregulation in a chitin-free experimental system (6Zhu Z. Zheng T. Homer R.J. Kim Y.K. Chen N.Y. Cohn L. Hamid Q. Elias J.A. Science. 2004; 304: 1678-1682Crossref PubMed Scopus (697) Google Scholar). These studies also demonstrated that epithelial cells store AMCase in intracellular granules and secrete the enzyme into their local microenvironment (7Homer R.J. Zhu Z. Cohn L. Lee C.G. White W.I. Chen S. Elias J.A. Am. J. Physiol. 2006; 291: L502-L511Crossref PubMed Scopus (72) Google Scholar). However, the pathways regulating the secretion of AMCase and the effects that it has after secretion have not been adequately defined. T helper cell type 2 acidic mammalian chitinase epidermal growth factor receptor transforming growth factor-α interleukin epidermal growth factor receptor extracellular signal-regulated kinase phosphate-buffered saline wild type ovalbumin. Epidermal growth factor receptor (EGFR) activation regulates epithelial cell activation, differentiation, proliferation, and survival (8Amishima M. Munakata M. Nasuhara Y. Sato A. Takahashi T. Homma Y. Kawakami Y. Am. J. Respir. Crit. Care Med. 1998; 157: 1907-1912Crossref PubMed Scopus (205) Google Scholar, 9Puddicombe S.M. Polosa R. Richter A. Krishna M.T. Howarth P.H. Holgate S.T. Davies D.E. FASEB J. 2000; 14: 1362-1374Crossref PubMed Google Scholar, 10Takeyama K. Dabbagh K. Lee H.M. Agusti C. Lausier J.A. Ueki I.F. Grattan K.M. Nadel J.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3081-3086Crossref PubMed Scopus (517) Google Scholar). EGFR can be activated by a variety of cell surface-bound EGFR ligands like TGF-α and amphiregulin (11Wells A. Int. J. Biochem. Cell Biol. 1999; 31: 637-643Crossref PubMed Scopus (899) Google Scholar, 12Davies D.E. Polosa R. Puddicombe S.M. Richter A. Holgate S.T. Allergy. 1999; 54: 771-783PubMed Google Scholar). Most frequently, the availability of these ligands is regulated via ectodomain shedding and receptor transactivation by specific metalloproteinases (13Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar, 14Lee D.C. Sunnarborg S.W. Hinkle C.L. Myers T.J. Stevenson M.Y. Russell W.E. Castner B.J. Gerhart M.J. Paxton R.J. Black R.A. Chang A. Jackson L.F. Ann. N. Y. Acad. Sci. 2003; 995: 22-38Crossref PubMed Scopus (154) Google Scholar), such as the ADAM17 (a membrane disintegrin and metallopeptidase domain 17), also called TACE (tumor necrosis factor-α-converting enzyme) (14Lee D.C. Sunnarborg S.W. Hinkle C.L. Myers T.J. Stevenson M.Y. Russell W.E. Castner B.J. Gerhart M.J. Paxton R.J. Black R.A. Chang A. Jackson L.F. Ann. N. Y. Acad. Sci. 2003; 995: 22-38Crossref PubMed Scopus (154) Google Scholar). Several lines of evidence have linked ADAM17/EGFR and asthma, including animal studies that demonstrated that many of the epithelial alterations at sites of Th2 inflammation can be ameliorated by EGFR inhibition (10Takeyama K. Dabbagh K. Lee H.M. Agusti C. Lausier J.A. Ueki I.F. Grattan K.M. Nadel J.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3081-3086Crossref PubMed Scopus (517) Google Scholar) and human studies that documented the exaggerated EGFR activation in tissues from patients with asthma (16Takeyama K. Fahy J.V. Nadel J.A. Am. J. Respir. Crit. Care Med. 2001; 163: 511-516Crossref PubMed Scopus (188) Google Scholar). A number of studies have also linked epithelial ADAM17 and EGFR signaling to IL-13. These studies demonstrated that IL-13 induces mucus metaplasia via an EGFR-dependent pathway (17Shim J.J. Dabbagh K. Ueki I.F. Dao-Pick T. Burgel P.R. Takeyama K. Tam D.C. Nadel J.A. Am. J. Physiol. 2001; 280: L134-L140Crossref PubMed Google Scholar), ADAM17 plays a critical role in the regulation of MUC5AC mucin expression in cultured human airway epithelial cells (18Shao M.X. Ueki I.F. Nadel J.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11618-11623Crossref PubMed Scopus (186) Google Scholar), and IL-13 induces the proliferation of normal human bronchial epithelial cells via ADAM17-induced ectodomain shedding of TGF-α (19Booth B.W. Sandifer T. Martin E.L. Martin L.D. Respir. Res. 2007; 8: 51-59Crossref PubMed Scopus (39) Google Scholar). Given the important roles of ADAM17 and EGFR signaling in IL-13-mediated activation of epithelial cells and allergic asthma, we hypothesized that a ADAM17/EGFR pathway contributes to the secretion of AMCase by lung epithelial cells. We also hypothesized that secreted AMCase feeds back to regulate epithelial cell function. To test these hypotheses, studies were undertaken to define the interactions of AMCase and EGFR and the role of the EGFR pathway in epithelial AMCase secretion. The effector responses of secreted AMCase were also evaluated. These studies demonstrate that respiratory epithelial cells secrete AMCase via a ADAM17/EGFR/Ras-dependent pathway. They also highlight the ability of AMCase to stimulate epithelial chemokine elaboration. Reagents—Cell culture media and fetal bovine serum were purchased from Invitrogen. Restriction endonucleases and other DNA-modifying enzymes were obtained from New England Biolabs (Beverly, MA). Oligonucleotides were synthesized by IDT, Inc. (Coralville, IA) or the Yale University Keck facility center. cDNAs were purchased from Clontech. PCR kits for gene amplification or cloning were obtained from Stratagene (La Jolla, CA). The immunoprecipitation kit was from Millipore (Billerica, MA). Staurosporine, the PI3 kinase inhibitor wortmannin, and the Ras inhibitor manumycin A (a farnesyltransferase inhibitor of Ras) were from Sigma. The pharmacological inhibitors PD153035 (a kinase inhibitor that specifically blocks the ATP binding site and inactivates EGFR signaling) and AG1478 (a small molecule EGFR tyrosine kinase inhibitor) were purchased from BIOSOURCE (Camarillo, CA). The TAPI-1 and -2 (TNFα-processing inhibitor-1/2, ADAM17-specific inhibitors) were from Peptides International (Louisville, KY). The ERK inhibitor PD98059 was bought from Calbiochem. The primary anti-human polycloncal and the rabbit anti-mouse monocloncal AMCase antibodies were generated using phage display technology and were provided by Medimmune (Gaithersburg, MD). Recombinant human AMCase was from Medimmune (Gaithersburg, MD). Mouse monoclonal antibody reactive to β-tubulin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary APC-labeled antibodies were from BD Biosciences. A primary anti-EGFR was from Abgent (San Diego, CA). Anti-human ADAM17 monoclonal antibody was purchased from R&D Systems (Minneapolis, MN) and employed as reported previously (20Walcheck B. Herrera A.H. St Hill C. Mattila P.E. Whitney A.R. Deleo F.R. Eur. J. Immunol. 2006; 36: 968-976Crossref PubMed Scopus (49) Google Scholar). Secondary Pe-Cy7- and APC-Cy7-labeled antibodies were from Santa Cruz Biotechnology. An anti-mouse CD31 fluorescein isothiocyanate antibody and the corresponding isotype control were from BD Biosciences. Anti-mouse pancytokeratin PE antibody was from Abcam. Anti-mouse CD45-PcP was from BD Biosciences. Flow Cytometry—Whole lung cell suspensions were obtained by a modified method according to Rice et al. (48Rice W.R. Conkright J.J. Na C.L. Ikegami M. Shannon J.M. Weaver T.E. Am. J. Physiol. 2002; 283: L256-L264Crossref PubMed Scopus (142) Google Scholar). In brief, lung tissue was digested using dispase (5 mg/ml; Stem Cell Technologies), collagenase (0.04%; Sigma), and 100 units/ml DNase (Sigma). Whole lung suspension cells underwent several centrifugations (10 min, 300–1000 g) and hemolysis (precooled hemolysis solution containing 11 mm KHCO3, 152 mm NH4Cl; washing for 5 min, 400 g at 4 °C), were then strained through progressively smaller cell strainers (100–20 μm) and nylon gauze, and were finally resuspended in fluorescence-activated cell sorting buffer (PBS, 2% bovine serum albumin, 2% fetal calf serum) supplemented with 10 units/ml DNase I. Macrophages were depleted from the lung cell suspensions by repeated means of adhesion to plastic plates at 37 °C. For cell surface staining, cells were treated for 30 min at 4 °C with appropriate combinations of specific antibodies for surface staining of CD45, CD31, EGFR, or ADAM17. The corresponding isotype controls or secondary antibodies only were stained in parallel. For intracellular staining, cells were fixed with 0.5 ml of ice-cold 2% paraformaldehyde and permeabilized using 0.5% saponin (Sigma) prior to antibody staining with anti-AMCase or anti-pancytokeratin or the respective isotype controls. Lung epithelial cells were characterized according to the following gating algorithm. Within digested and strained red cell and macrophage-depleted whole lung cell suspensions, nondebris cells were gated, and CD45+ cells were further excluded. Within CD45- negative lung cells, CD31+ endothelial cells were excluded. Within the CD45-CD31- lung cell population, cells positive for intracellular pancytokeratin were considered as epithelial cells according to a modified method, as described previously (21Schutte B. Tinnemans M.M. Pijpers G.F. Lenders M.H. Ramaekers F.C. Cytometry. 1995; 21: 177-186Crossref PubMed Scopus (29) Google Scholar, 22Rochat T.R. Casale J.M. Hunninghake G.W. J. Lab. Clin. Med. 1988; 112: 418-425PubMed Google Scholar). For co-expression studies of intracellular proteins (AMCase) with extracellular receptors (ADAM17 and EGFR), lung cells were first surface-stained with anti-EGFR or anti-ADAM17, were permeabilized, and then underwent staining for intracellular AMCase. Saturating concentrations of the antibodies were used, as determined by titration experiments prior to the study. At least 10,000 cells/sample were analyzed. All antibodies and fluorescence-activated cell sorting reagents were from BD Biosciences except when otherwise indicated. Isotype controls were subtracted from the respective specific antibody expression, and the results are reported as mean fluorescence intensity. Calculations were performed with Cell Quest analysis software (BD Biosciences). All experiments were performed in triplicate. IL-13-overexpressing Mice—C57BL/6 wild type (WT) were obtained from the Jackson Laboratories (Bar Harbor, ME). CC10-rtTA-IL-13 transgenic mice were generated in our laboratory (23Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese Jr., R.J. Chapman Jr., H.A. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar), bred onto a C57BL/6 background, and used in these studies. These mice utilize the Clara cell 10-kDa protein (CC10) promoter and the reverse tetracycline transactivator (rtTA) to target IL-13 to the lung in a doxycycline-inducible manner. These animals have very low to undetectable levels of IL-13 in their lungs at base line and 1.9–2.1 ng/ml quantities after transgene induction by adding doxycycline for 4 weeks to the animals' drinking water. When CC10-IL-13 mice were being evaluated, Tg (-) littermate animals were used as controls. These studies were approved by the Yale University School of Medicine Institutional Animal Care and Use Committee. OVA Sensitization and Challenge—OVA sensitization and challenge were accomplished using a modification of the protocols previously described by our laboratory (23Zheng T. Zhu Z. Wang Z. Homer R.J. Ma B. Riese Jr., R.J. Chapman Jr., H.A. Shapiro S.D. Elias J.A. J. Clin. Invest. 2000; 106: 1081-1093Crossref PubMed Scopus (546) Google Scholar). In brief, 6–8-week-old WT mice were received injections containing 20 μg of chicken OVA (Sigma) complexed to alum (Resorptar, Indergen, New York, NY) or alum alone. This process was repeated 5 days later. After an additional 7 days, the animals received three aerosol challenges (40 min a day, 3 days) with 1% OVA (w/v) in endotoxin-free PBS or PBS alone. The aerosol was generated in a NE-U07 ultrasonic nebulizer (Omron Health Care, Vernon Hills, IL). The mice were sacrificed 24, 48, or 72 h after aerosol exposure. Expression of Recombinant AMCase and EGFR—Human AMCase full-length cDNA plasmid constructed in expression vector pcDNA3.1 was obtained from MedImmune (Gaithersburg, MD). Human AMCase cDNA was amplified using PCR with the following primers: hAMCase-L-upper, 5′-ATG GAG GCC GAA TTC ATG GTT TCT ACT CCT GAG AAC-3′; hAMCase-L-lower, 5′-ATC TGC AGA ATT CCA CAT TGC CCA GTT GCA GCA ATC-3′. The wild-type human full-length EGFR cloned into the retroviral LXSN vector was a gift from David Stern (Yale University, New Haven, CT). The plasmid pCMV-β-Gal (American Type Culture Collection, Manassas, VA) was used to monitor transfection efficiency. A549 cells were incubated for 6 h with vector DNA mixtures that did not contain pcDNA 3.1 or contained AMCase or EGFR inserts. After incubation, the cells were washed and incubated for an additional 18 or 42 h in complete medium for immunoprecipitation assay and in Opti-MEM medium for bioactivity assay. Under these conditions, the transfection efficiency was found to be greater than 90% as determined by a β-galactosidase assay. The samples were centrifuged at 14,000 rpm in an Eppendorff microcentrifuge at 4 °C for 5 min, and the clear supernatants were saved. The presence of AMCase was verified by Western blotting. The activity of expressed AMCase was verified by an enzymatic assay as described below in detail. Protein Extraction and Western Blot Analysis—Whole lung and cell monolayer lysates were evaluated by Western blotting. Lung lysates were prepared using lysis buffer as previously described. The cell monolayers were washed twice with ice-cold PBS containing 1 mm sodium orthovanadate and 1 mm sodium fluoride and lysed with lysis buffer (15 mm Hepes, pH 7.9, 10% glycerol, 0.5% Nonidet P-40, 250 mm NaCl, 0.1 mm EDTA, 1 mm sodium orthovanadate, 1 mm sodium fluoride, 10 mm dithiothreitol, and one tablet of complete miniprotease inhibitor mixture/10 ml of lysis buffer). The lysates were then clarified by centrifugation at 10,000 × g for 15 min, and supernatant protein concentrations were determined with a Bio-Rad assay kit. The samples were then mixed with an equal volume of 2× SDS-PAGE sample buffer (100 mm Tris-Cl, pH 6.8, 200 mm dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% glycerol) and heated in a boiling water bath, and equal amounts were loaded onto 12% SDS-polyacrylamide gels (Bio-Rad) and transferred to Immune-Blot polyvinylidene difluoride membrane (Bio-Rad). After transfer, the membranes were blocked for 1 h in nonfat dried milk, rinsed, incubated with the appropriate primary antibodies for 1.5 h at room temperature or overnight at 4 °C, washed, incubated with secondary antibody (diluted 1:1000–1:2000) for 1.5 h at room temperature, and washed in 0.05% Tween 20. Immunoreactive proteins were visualized using 20× LumiGLO Reagent and 20× peroxide according to the manufacturer's instructions (Cell Signaling Technology Inc., Beverly, MA). The membranes were exposed to BioMax MR film (Eastman Kodak Co.). Immunoprecipitation—A549 cells (2 × 106/10-cm diameter dish) were transfected with 24 μg of DNA with 60 μl of Lipofectamine 2000. 48 h later, the cell monolayers were washed twice with ice-cold PBS containing 1 mm sodium orthovanadate and 1 mm sodium fluoride and lysed with lysis buffer mentioned above. The lysate was clarified by centrifugation for 10 min at 4 °C. AMCase and associated proteins were immunoprecipitated with rabbit anti-AMCase polyclonal antibody or anti-EGFR (Invitrogen), respectively, using the Catch and Release version 2.0 reversible immunoprecipitation system (Millipore). The precipitate was transferred to a polyvinylidene difluoride membrane and probed with monoclonal antibody against EGFR or AMCase followed by chemiluminescence detection. Parallel protein immunoblots of each sample were performed to confirm the expected expression of EGFR or AMCase constructs in the cells. Immunofluorescence Staining and Confocal Microscopy— Immunohistochemistry of AMCase was performed as described by our laboratory previously (7Homer R.J. Zhu Z. Cohn L. Lee C.G. White W.I. Chen S. Elias J.A. Am. J. Physiol. 2006; 291: L502-L511Crossref PubMed Scopus (72) Google Scholar), using a polyclonal rabbit anti-human AMCase or monocloncal rabbit anti-mouse antibody developed by MedImmune (Gaithersburg, MD) (171.204), with specificity for AMCase. The antibody was applied to the lung sections at a 1:100 dilution. To verify the specificity of the reactions, the rabbit antibody was incubated with AMCase-specific peptide (amino acids 428–446) at a 1:1 ratio for 2 h before being applied to the tissues. The mouse monoclonal antibody specificity was determined by ELISA against other murine chitinase family members (chitotriosidase, Ym1, Ym2, and YKL-40) and by comparison of the staining pattern with the rabbit antibody as described previously by our group (7Homer R.J. Zhu Z. Cohn L. Lee C.G. White W.I. Chen S. Elias J.A. Am. J. Physiol. 2006; 291: L502-L511Crossref PubMed Scopus (72) Google Scholar). In all cases, BD Pharmingen™ Retrievagen A (pH 6) antigen retrieval solution was used for 20 min. The avidin/biotin blocking kit (Vector Laboratories, Inc., Burlingame, CA) was selected to treat the section for two-color staining of lung tissue. For two-color fluorescence, secondary reagents included anti-mouse or anti-rabbit, either biotinylated or directly conjugated with Alexa-488 (Molecular Probes) or anti-rabbit Cy3 (Sigma). Tissues were mounted using VECTASHIELD mounting medium for fluorescence (Vector Laboratories, Inc., Burlingame, CA). For combined AMCase-EGFR staining, streptavidin Alexa-488 (green dye) was combined with Cy3 (red dye). Confocal microscopy was performed with a Zeiss LSM 510 META system with an optical thickness of either 5.0 or 1.0 μm. Chitinase Bioactivity—The chitinase bioactivity in cell culture supernatants and cell lysates was determined using a fluorogenic substrate as described previously (24Spindler K.D. Spindler-Barth M. EXS. 1999; 87: 201-209PubMed Google Scholar). Briefly, 50 μl of each sample was mixed with 30 μl of citrate/phosphate buffer (0.1 and 0.2 m, respectively), pH 5.2, and 20 μl of 0.5 mg/ml substrate 4-methylumbelliferyl-d-N,N′-diacetylchitobioside (Sigma) at a final concentration of 0.17 mm. The samples were incubated at 37 °C for varying amounts of time, and the reactions were stopped by adding 1 ml of stop solution (0.3 m glycine/NaOH buffer, pH 10.6). The fluorescence intensity of released 4-methylumbelliferone was measured with a fluorometer (excitation 350 nm and emission 450 nm). A standard curve was generated using serially diluted 4-methylumbelliferone (Sigma). Chitinase extracted from Serratia marcescens (Sigma) was used as a positive control. Cytokine and Chemokine ELISA—Immunosandwich enzyme-linked immunosorbent assay kits for IL-6, MCP-1/CCL2, IL-8/CXCL8, and TARC/CCL17 were used according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). Statistics—All data were initially checked for normal/parametric distribution (Kolmogorov-Smirnov test). If parametric distribution was found, analysis of variance was applied to screen for differences among at least three groups. To compare two individual groups, Student's t test was applied. If nonparametric distribution was found, the Kruskal-Wallis test was applied to screen for differences among at least three groups, followed by the Mann-Whitney U test (Wilcoxon rank sum test) to compare two individual groups. Statistical analyses were performed using Prism 4.0 (Graph Pad Software) and STATA version 8.2 for Windows (STATA Corp.) according to the approach described in Ref. 25Motulsky H. Intuitive Biostatistics. Oxford University Press, New York1995Google Scholar. AMCase, EGFR, and ADAM17 Are Induced and Co-localize in Vivo at Sites of Th2 and Th2-Cytokine-mediated Airway Inflammation—To characterize and quantify intracellular AMCase expression in lung epithelial cells in vivo, we established a flow cytometric epithelial evaluation method based on light scatter parameters, negative expression of CD45, negative expression of CD31, and positive expression of cytokeratin (Fig. 1). Using this method, we successfully compared the levels of intracellular AMCase in lung epithelial cells from control mice, mice sensitized and challenged with OVA, and IL-13-overexpressing transgenic animals. These studies demonstrated a significant increase in intracellular AMCase in both of the modeling systems (Fig. 2, a–d). The increase in intracellular AMCase in these Th2 inflammation/asthma models was paralleled by an increase of total EGFR in lung lysates (Fig. 3a) as well as specific EGFR surface expression on lung epithelial cells as analyzed by immunohistochemistry (Fig. 3b) and flow cytometry (Fig. 3, c and d). Using confocal microscopy, AMCase and EGFR were found to co-localize in lung epithelial cells of lungs of IL-13 transgenic and OVA-sensitized and -challenged mice (Fig. 3e) (data not shown).FIGURE 2Th2 airway inflammation increases AMCase expression in lung epithelial cells. Intracellular AMCase was stained in permeabilized CD45-CD31-cytokeratin+ lung epithelial cells in WT and IL-13-overexpressing transgenic mice (IL 13 TG) (a and c), PBS-sensitized and challenged mice (PBS), and OVA-sensitized and challenged mice (OVA) (b). Bars, means ± S.D. (*, p < 0.05; Student's t test). c, a representative histogram of intracellular AMCase expression in CD45-CD31-cytokeratin+ cells. Green line, isotype (ISO) control; red line, AMCase expression in CD45-CD31-cytokeratin+ cells from WT mice; filled blue area, AMCase expression in CD45-CD31-cytokeratin+ cells from IL-13 Tg mice. d, a representative histogram of intracellular AMCase expression in CD45-CD31-cytokeratin+ cells. Green line, isotype control; red line, AMCase expression in CD45-CD31-cytokeratin+ cells from WT mice; filled blue area, AMCase expression in CD45-CD31-cytokeratin+ cells from OVA-sensitized and challenged (OVA) mice. MFI, mean fluorescence intensity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Th2 airway inflammation increases EGFR and ADAM17 expression in lung epithelial cells. a, immunoblotting of EGFR expression in whole lung cell lysates in PBS-sensitized and challenged (PBS) and OVA-sensitized and challenged mice (OVA) (top). WT and IL-13-overexpressing transgenic mice (IL-13 tg) are seen in the lower panel. b, EGFR expression in lung epithelial cells using immunohistochemistry in WT and IL-13-overexpressing transgenic mice. In the upper panel an isotype control IgG antibody was used instead of the specific anti-EGFR antibody. c, EGFR surface expression on CD45-CD31-cytokeratin+ cells in WT and IL-13-overexpressing transgenic mice, PBS-sensitized and -challenged mice, and OVA-sensitized and -challenged mice. Bars, means ± S.D. (*, p < 0.05; Student's t test). d, representative histogram of EGFR surface expression on CD45-CD31-cytokeratin+ cells. The left panel shows EGFR expression in lungs from IL-13-overexpressing transgenic mice, and the right panel shows expression in lungs from OVA-sensitized and -challenged (OVA) mice. Green line, isotype (ISO) control; filled blue area, EGFR expression on CD45-CD31-cytokeratin+ cells of WT mice; red line, EGFR expression on CD45-CD31-cytokeratin+ cells from IL-13-overexpressing transgenic mice. e, AMCase (Alexa-488, green dye) and EGFR (Cy3, red dye) were localized in tissues from IL-13-overexpressing transgenic mice. Confocal microscopy was performed with a Zeiss LSM 510 META system. Co-localization of AMCase and EGFR after merging was observed as a yellow color with confocal microscopy. f, ADAM17 surface expression was evaluated on CD45-CD31-cytokeratin+ lung epithelial cells from WT and IL-13-overexpressing transgenic (left) mice, PBS-sensitized and challenged mice, and OVA-sensitized and challenged mice (right) (p < 0.05; S" @default.
• W2003443332 created "2016-06-24" @default.
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• W2003443332 creator A5024105936 @default.
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• W2003443332 date "2008-11-01" @default.
• W2003443332 modified "2023-09-30" @default.
• W2003443332 title "Acidic Mammalian Chitinase Is Secreted via an ADAM17/Epidermal Growth Factor Receptor-dependent Pathway and Stimulates Chemokine Production by Pulmonary Epithelial Cells" @default.
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