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Extracellular Superoxide Dismutase in Macrophages Augments Bacterial Killing by Promoting Phagocytosis

2011; Elsevier BV; Volume: 178; Issue: 6 Linguagem: Inglês

10.1016/j.ajpath.2011.02.007

ISSN

1525-2191

Autores

Michelle L. Manni, Lauren Tomai, Callie A. Norris, L. Michael Thomas, Eric E. Kelley, Russell D. Salter, James D. Crapo, Ling‐Yi Chang, Simon C. Watkins, Jon D. Piganelli, Tim D. Oury,

Tópico(s)

Cardiac Arrest and Resuscitation

Resumo

Extracellular superoxide dismutase (EC-SOD) is abundant in the lung and limits inflammation and injury in response to many pulmonary insults. To test the hypothesis that EC-SOD has an important role in bacterial infections, wild-type and EC-SOD knockout (KO) mice were infected with Escherichia coli to induce pneumonia. Although mice in the EC-SOD KO group demonstrated greater pulmonary inflammation than did wild-type mice, there was less clearance of bacteria from their lungs after infection. Macrophages and neutrophils express EC-SOD; however, its function and subcellular localization in these inflammatory cells is unclear. In the present study, immunogold electron microscopy revealed EC-SOD in membrane-bound vesicles of phagocytes. These findings suggest that inflammatory cell EC-SOD may have a role in antibacterial defense. To test this hypothesis, phagocytes from wild-type and EC-SOD KO mice were evaluated. Although macrophages lacking EC-SOD produced more reactive oxygen species than did cells expressing EC-SOD after stimulation, they demonstrated significantly impaired phagocytosis and killing of bacteria. Overall, this suggests that EC-SOD facilitates clearance of bacteria and limits inflammation in response to infection by promoting bacterial phagocytosis. Extracellular superoxide dismutase (EC-SOD) is abundant in the lung and limits inflammation and injury in response to many pulmonary insults. To test the hypothesis that EC-SOD has an important role in bacterial infections, wild-type and EC-SOD knockout (KO) mice were infected with Escherichia coli to induce pneumonia. Although mice in the EC-SOD KO group demonstrated greater pulmonary inflammation than did wild-type mice, there was less clearance of bacteria from their lungs after infection. Macrophages and neutrophils express EC-SOD; however, its function and subcellular localization in these inflammatory cells is unclear. In the present study, immunogold electron microscopy revealed EC-SOD in membrane-bound vesicles of phagocytes. These findings suggest that inflammatory cell EC-SOD may have a role in antibacterial defense. To test this hypothesis, phagocytes from wild-type and EC-SOD KO mice were evaluated. Although macrophages lacking EC-SOD produced more reactive oxygen species than did cells expressing EC-SOD after stimulation, they demonstrated significantly impaired phagocytosis and killing of bacteria. Overall, this suggests that EC-SOD facilitates clearance of bacteria and limits inflammation in response to infection by promoting bacterial phagocytosis. Acute infection of the lower respiratory tract is the leading infectious cause of premature death, with a greater disease burden than cancer.1Mizgerd J.P. Lung infection: a public health priority.PLoS Med/Public Library of Science. 2006; 3: e76Google Scholar, 2Mizgerd J.P. Mizgerd J.P. Acute lower respiratory tract infection.N Engl J Med. 2008; 358: 716-727Crossref PubMed Scopus (330) Google Scholar, 3Mizgerd J.P. Skerrett S.J. Animal models of human pneumonia.Am J Physiol Lung Cell Mol Physiol. 2008; 294: L387-L398Crossref PubMed Scopus (110) Google Scholar In the lung, alveolar macrophages and neutrophils are phagocytic inflammatory cells that have a central role in the elimination of bacterial infections. As the first line of defense, macrophages and neutrophils produce potent molecules such as reactive oxygen species (ROS) and proteases in an attempt to eliminate microbes. While this antimicrobial arsenal is beneficial for eradicating the infectious organisms, these toxic agents can directly cause pulmonary damage and contribute to complications such as acute respiratory distress syndrome.4Windsor A.C. Mullen P.G. Fowler A.A. Sugerman H.J. Role of the neutrophil in adult respiratory distress syndrome.Br J Surg. 1993; 80: 10-17Crossref PubMed Scopus (233) Google Scholar, 5Sibille Y. Marchandise F.X. Pulmonary immune cells in health and disease: polymorphonuclear neutrophils.Eur Respir J. 1993; 6: 1529-1543PubMed Google Scholar, 6Hashimoto S. Okayama Y. Shime N. Kimura A. Funakoshi Y. Kawabata K. Ishizaka A. Amaya F. Neutrophil elastase activity in acute lung injury and respiratory distress syndrome.Respirology. 2008; 13: 581-584Crossref PubMed Scopus (47) Google Scholar Endogenous antioxidant enzymes are normal cellular defenses that can protect against ROS-induced tissue injury. Extracellular superoxide dismutase (EC-SOD) is a 135-kDa antioxidant enzyme that scavenges the free radical superoxide.7Fattman C.L. Schaefer L.M. Oury T.D. Extracellular superoxide dismutase in biology and medicine.Free Radic Biol Med. 2003; 35: 236-256Crossref PubMed Scopus (531) Google Scholar This isozyme of the superoxide dismutase family is highly expressed in the lung and arteries and is bound to the extracellular matrix via its positively charged heparin/matrix-binding domain.7Fattman C.L. Schaefer L.M. Oury T.D. Extracellular superoxide dismutase in biology and medicine.Free Radic Biol Med. 2003; 35: 236-256Crossref PubMed Scopus (531) Google Scholar, 8Oury T.D. Day B.J. Crapo J.D. Extracellular superoxide dismutase in vessels and airways of humans and baboons.Free Radic Biol Med. 1996; 20: 957-965Crossref PubMed Scopus (209) Google Scholar, 9Oury T.D. Crapo J.D. Valnickova Z. Enghild J.J. Human extracellular superoxide dismutase is a tetramer composed of two disulphide-linked dimers: a simplified, high-yield purification of extracellular superoxide dismutase.Biochem J. 1996; 317: 51-57PubMed Google Scholar, 10Fattman C.L. Enghild J.J. Crapo J.D. Schaefer L.M. Valnickova Z. Oury T.D. Purification and characterization of extracellular superoxide dismutase in mouse lung.Biochem Biophys Res Commun. 2000; 275: 542-548Crossref PubMed Scopus (56) Google Scholar EC-SOD acts as both an anti-inflammatory and antifibrotic agent in a number of pulmonary diseases including bleomycin- and asbestos-induced pulmonary fibrosis,11Fattman C.L. Tan R.J. Tobolewski J.M. Oury T.D. Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase.Free Radic Biol Med. 2006; 40: 601-607Crossref PubMed Scopus (77) Google Scholar, 12Tan R.J. Fattman C.L. Watkins S.C. Oury T.D. Redistribution of pulmonary EC-SOD after exposure to asbestos.J Appl Physiol. 2004; 97: 2006-2013Crossref PubMed Scopus (68) Google Scholar, 13Fattman C.L. Chang L.Y. Termin T.A. Petersen L. Enghild J.J. Oury T.D. Enhanced bleomycin-induced pulmonary damage in mice lacking extracellular superoxide dismutase.Free Radic Biol Med. 2003; 35: 763-771Crossref PubMed Scopus (104) Google Scholar, 14Bowler R.P. Nicks M. Warnick K. Crapo J.D. Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis.Am J Physiol Lung Cell Mol Physiol. 2002; 282: L719-L726Crossref PubMed Scopus (13) Google Scholar hyperoxia,15Carlsson L.M. Jonsson J. Edlund T. Marklund S.L. Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia.Proc Natl Acad Sci USA. 1995; 92: 6264-6268Crossref PubMed Scopus (406) Google Scholar, 16Folz R.J. Abushamaa A.M. Suliman H.B. Extracellular superoxide dismutase in the airways of transgenic mice reduces inflammation and attenuates lung toxicity following hyperoxia.J Clin Invest. 1999; 103: 1055-1066Crossref PubMed Scopus (255) Google Scholar, 17Oury T.D. Schaefer L.M. Fattman C.L. Choi A. Weck K.E. Watkins S.C. Depletion of pulmonary EC-SOD after exposure to hyperoxia.Am J Physiol Lung Cell Mol Physiol. 2002; 283: L777-L784PubMed Google Scholar lipopolysaccharide-induced inflammation,18Bowler R.P. Nicks M. Tran K. Tanner G. Chang L.Y. Young S.K. Worthen G.S. Extracellular superoxide dismutase attenuates lipopolysaccharide-induced neutrophilic inflammation.Am J Respir Cell Mol Biol. 2004; 31: 432-439Crossref PubMed Scopus (92) Google Scholar and pulmonary infection.19Tan R.J. Lee J.S. Manni M.L. Fattman C.L. Tobolewski J.M. Zheng M. Kolls J.K. Martin T.R. Oury T.D. Inflammatory cells as a source of airspace extracellular superoxide dismutase after pulmonary injury.Am J Respir Cell Mol Biol. 2006; 34: 226-232Crossref PubMed Scopus (34) Google Scholar, 20Suliman H.B. Ryan L.K. Bishop L. Folz R.J. Prevention of influenza-induced lung injury in mice overexpressing extracellular superoxide dismutase.Am J Physiol Lung Cell Mol Physiol. 2001; 280: L69-L78PubMed Google Scholar One mechanism by which EC-SOD inhibits inflammation is directly binding to and inhibiting oxidative fragmentation of several components in the extracellular matrix including collagen, heparan sulfate, and hyaluronan after interstitial lung injury.11Fattman C.L. Tan R.J. Tobolewski J.M. Oury T.D. Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase.Free Radic Biol Med. 2006; 40: 601-607Crossref PubMed Scopus (77) Google Scholar, 21Gao F. Koenitzer J.R. Tobolewski J.M. Jiang D. Liang J. Noble P.W. Oury T.D. Extracellular superoxide dismutase inhibits inflammation by preventing oxidative fragmentation of hyaluronan.J Biol Chem. 2008; 283: 6058-6066Crossref PubMed Scopus (151) Google Scholar, 22Kliment C.R. Tobolewski J.M. Manni M.L. Tan R.J. Enghild J. Oury T.D. Extracellular superoxide dismutase protects against matrix degradation of heparan sulfate in the lung.Antioxid Redox Signal. 2008; 10: 261-268Crossref PubMed Scopus (77) Google Scholar, 23Kliment C.R. Englert J.M. Gochuico B.R. Yu G. Kaminski N. Rosas I. Oury T.D. Oxidative stress alters syndecan-1 distribution in lungs with pulmonary fibrosis.J Biol Chem. 2009; 284: 3537-3545Crossref PubMed Scopus (106) Google Scholar, 24Petersen S.V. Oury T.D. Ostergaard L. Valnickova Z. Wegrzyn J. Thogersen I.B. Jacobsen C. Bowler R.P. Fattman C.L. Crapo J.D. Enghild J.J. Extracellular superoxide dismutase (EC-SOD) binds to type I collagen and protects against oxidative fragmentation.J Biol Chem. 2004; 279: 13705-13710Crossref PubMed Scopus (152) Google Scholar The importance of endogenous pulmonary EC-SOD was recently highlighted in a study that demonstrated that acute loss of EC-SOD resulted in 85% mortality secondary to the spontaneous development of acute respiratory distress syndrome.25Gongora M.C. Lob H.E. Landmesser U. Guzik T.J. Martin W.D. Ozumi K. Wall S.M. Wilson D.S. Murthy N. Gravanis M. Fukai T. Harrison D.G. Loss of extracellular superoxide dismutase leads to acute lung damage in the presence of ambient air: a potential mechanism underlying adult respiratory distress syndrome.Am J Pathol. 2008; 173: 915-926Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar This indicates that EC-SOD is essential for protecting the lungs against inflammation and injury even in ambient air. In addition to its localization on matrices and in extracellular fluids, EC-SOD is also present intracellularly in neutrophils and macrophages.13Fattman C.L. Chang L.Y. Termin T.A. Petersen L. Enghild J.J. Oury T.D. Enhanced bleomycin-induced pulmonary damage in mice lacking extracellular superoxide dismutase.Free Radic Biol Med. 2003; 35: 763-771Crossref PubMed Scopus (104) Google Scholar, 19Tan R.J. Lee J.S. Manni M.L. Fattman C.L. Tobolewski J.M. Zheng M. Kolls J.K. Martin T.R. Oury T.D. Inflammatory cells as a source of airspace extracellular superoxide dismutase after pulmonary injury.Am J Respir Cell Mol Biol. 2006; 34: 226-232Crossref PubMed Scopus (34) Google Scholar, 26Loenders B. Van Mechelen E. Nicolai S. Buyssens N. Van Osselaer N. Jorens P.G. Willems J. Herman A.G. Slegers H. Localization of extracellular superoxide dismutase in rat lung: neutrophils and macrophages as carriers of the enzyme.Free Radic Biol Med. 1998; 24: 1097-1106Crossref PubMed Scopus (44) Google Scholar However, the localization and role of EC-SOD in these inflammatory cells have not been investigated. Few studies have investigated the role of this potent antioxidant in bacterial infections; therefore, the present study investigated the response of wild-type and EC-SOD knockout (KO) mice to live Escherichia coli inoculation. To better understand the role of EC-SOD in infection, the subcellular localization and functional role of EC-SOD in inflammatory cells was also investigated. These findings are important for innate lung defense against E. coli in a murine model of pneumonia and possibly in other gram-negative bacterial infections. Nine-week-old sex-matched C57BL/6 mice (Taconic Farms, Inc., Hudson, NY) and EC-SOD KO mice (congenic in the C57BL/6 background13Fattman C.L. Chang L.Y. Termin T.A. Petersen L. Enghild J.J. Oury T.D. Enhanced bleomycin-induced pulmonary damage in mice lacking extracellular superoxide dismutase.Free Radic Biol Med. 2003; 35: 763-771Crossref PubMed Scopus (104) Google Scholar) were used for all animal studies. The animal studies were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Mice were intratracheally instilled with approximately 1 × 106 colony-forming units (CFUs) live E. coli (ATCC 25922; American Type Culture Collection, Manassas, VA) in 50 μL PBS27Ong E.S. Gao X.P. Xu N. Predescu D. Rahman A. Broman M.T. Jho D.H. Malik A.B. E. coli pneumonia induces CD18-independent airway neutrophil migration in the absence of increased lung vascular permeability.Am J Physiol Lung Cell Mol Physiol. 2003; 285: L879-L888PubMed Google Scholar and sacrificed immediately (0 hours) or at 6 or 24 hours after inoculation. The entire left lung was removed and placed in 1 mL sterile water for homogenization as previously described.19Tan R.J. Lee J.S. Manni M.L. Fattman C.L. Tobolewski J.M. Zheng M. Kolls J.K. Martin T.R. Oury T.D. Inflammatory cells as a source of airspace extracellular superoxide dismutase after pulmonary injury.Am J Respir Cell Mol Biol. 2006; 34: 226-232Crossref PubMed Scopus (34) Google Scholar Bronchoalveolar lavage fluid (BALF) was collected via instillation and recovery of 0.6 mL 0.9% saline solution from the right lung, and the right lung was then inflation-fixed with 10% formalin for histologic analyses. Cell counts with differential values in the BALF and cell differential counts were performed as described previously.12Tan R.J. Fattman C.L. Watkins S.C. Oury T.D. Redistribution of pulmonary EC-SOD after exposure to asbestos.J Appl Physiol. 2004; 97: 2006-2013Crossref PubMed Scopus (68) Google Scholar BALF supernatants were stored at −70°C until analyses. The left lung was homogenized as previously described,19Tan R.J. Lee J.S. Manni M.L. Fattman C.L. Tobolewski J.M. Zheng M. Kolls J.K. Martin T.R. Oury T.D. Inflammatory cells as a source of airspace extracellular superoxide dismutase after pulmonary injury.Am J Respir Cell Mol Biol. 2006; 34: 226-232Crossref PubMed Scopus (34) Google Scholar and the homogenates were separated into aliquots for quantitative cultures of bacteria, Western blot analysis, and myeloperoxidase assays. In brief, for Western blot analyses, buffer was added to an aliquot of lung homogenate to a final concentration of buffer containing 0.5% Triton X-100, 150 nmol/L NaCl, 15 mmol/L Tris, 1 mmol/L Ca Cl2, and 1 mmol/L MgCl2 (pH 7.4), incubated on ice for 30 minutes, and centrifuged at 10,000 × g for 20 minutes. The supernatants were stored at −80°C for later use. For myeloperoxidase activity measurements, the lung homogenate was added to a buffer to a final concentration of solution containing 50 mmol/L potassium phosphate (pH 6.0), 5 mmol/L EDTA, and 5% w/v hexadecyltrimethyl ammonium bromide. This mixture was then sonicated and centrifuged at 12,000 × g for 30 minutes. The supernatant was then stored at −80°C until use. Serial 10-fold dilutions of lung homogenates were cultured in Luria-Bertani agar using the pour plate method to determine the number of CFUs. Lung homogenates in 50 mmol/L potassium phosphate (pH 6.0), 5 mmol/L EDTA, and 5% w/v hexadecyltrimethyl ammonium bromide were assayed in triplicate at a ratio of 1:30 v/v in assay buffer [50 mmol/L potassium phosphate buffer (pH 6.0), 0.167 mg/mL o-dianisidine dihydrochloride, 0.0005% hydrogen peroxide, and 0.01% hexadecyltrimethylammonium bromide] in a 96-well plate. Immediately after addition of assay buffer, myeloperoxidase activity was monitored by measuring the absorbance at 460 nm over 2 minutes. Relative myeloperoxidase activity was calculated as the change in absorbance over time per left lung (dA/min/left lung). Macrophages derived from bone marrow were isolated and cultured from wild-type and EC-SOD KO mice as previously described.28Rhoades E.R. Orme I.M. Similar responses by macrophages from young and old mice infected with Mycobacterium tuberculosis.Mechanisms Ageing Dev. 1998; 106: 145-153Crossref PubMed Scopus (28) Google Scholar, 29Tse H.M. Josephy S.I. Chan E.D. Fouts D. Cooper A.M. Activation of the mitogen-activated protein kinase signaling pathway is instrumental in determining the ability of Mycobacterium avium to grow in murine macrophages.J Immunol. 2002; 168: 825-833PubMed Google Scholar Twenty-four hours before use, macrophages were washed and cultured with media lacking both antibiotics and L929 supplement. Peritoneal macrophages were isolated from the peritoneum via instillation and recovery of sterile PBS without calcium or magnesium. Alveolar macrophages were harvested via instillation and recovery of 0.8 mL cold sterile PBS from the lungs. Both cell types were allowed to adhere at 37°C in a humidified 5% CO2 atmosphere for 2 hours. Nonadherent cells were removed by means of gentle washing, and remaining cells were cultured overnight in media lacking both antibiotics and L929 supplement for experiments. The purity of the cell populations was verified at flow cytometry using macrophage marker phycoerythrin-conjugated anti-mouse F4/80 (Molecular Probes, Eugene, OR). For detection of EC-SOD, cell lysates were collected in ice-cold lysis buffer with protease inhibitors [1% Triton, 25 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L EDTA (pH 7.5) with 100 μmol/L 3,4-dichloroisocoumarin, 10 μmol/L E-64, and 1 mmol/L 1,10-phenanthroline]. EC-SOD in human phagocytes in normal lung was localized using electron microscopy with immunogold labeling as previously described.30Oury T.D. Chang L.Y. Marklund S.L. Day B.J. Crapo J.D. Immunocytochemical localization of extracellular superoxide dismutase in human lung.Lab Invest. 1994; 70: 889-898PubMed Google Scholar Western blot analysis of EC-SOD in BALF, cell lysate, or lung homogenates was performed as previously described,12Tan R.J. Fattman C.L. Watkins S.C. Oury T.D. Redistribution of pulmonary EC-SOD after exposure to asbestos.J Appl Physiol. 2004; 97: 2006-2013Crossref PubMed Scopus (68) Google Scholar, 23Kliment C.R. Englert J.M. Gochuico B.R. Yu G. Kaminski N. Rosas I. Oury T.D. Oxidative stress alters syndecan-1 distribution in lungs with pulmonary fibrosis.J Biol Chem. 2009; 284: 3537-3545Crossref PubMed Scopus (106) Google Scholar and standardized to β-actin as a loading control when appropriate. The EC-SOD antibodies used recognize both full-length and proteolyzed forms of this antioxidant enzyme. Electron paramagnetic resonance (EPR) spectroscopy was performed to determine the location and amount of oxidant production in wild-type and EC-SOD KO macrophages 10 minutes after stimulation with 15 μg/mL phorbol 12–myristate 13–acetate (PMA). Peritoneal macrophages isolated from wild-type and EC-SOD KO mice were resuspended (1 × 106 cells/mL) in Krebs HEPES buffer (pH 7.4). Identification of ROS production (superoxide, peroxynitrite, and other downstream single-electron reactive-oxygen intermediates) was accomplished by exposing the stimulated cells to either 50 μmol/L cell-permeable CMH (1-hydroxy-3-methoxy-carbonyl-2,2,5,5-tetramethylpyrrolidine) or 50 μmol/L cell-impermeable PPH (1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine) spin probes (Noxygen Science Transfer & Diagnostics GmbH, Elzach, Germany) and analyzed using a table-top EPR spectrometer (eScan; Bruker BioSpin, Billerica, MA).31Dikalov S.I. Li W. Mehranpour P. Wang S.S. Zafari A.M. Production of extracellular superoxide by human lymphoblast cell lines: comparison of electron spin resonance techniques and cytochrome c reduction assay.Biochem Pharmacol. 2007; 73: 972-980Crossref PubMed Scopus (58) Google Scholar, 32Dikalov S.I. Dikalova A.E. Bikineyeva A.T. Schmidt H.H. Harrison D.G. Griendling K.K. Distinct roles of Nox1 and Nox4 in basal and angiotensin II–stimulated superoxide and hydrogen peroxide production.Free Radic Biol Med. 2008; 45: 1340-1351Crossref PubMed Scopus (323) Google Scholar, 33Palazzolo-Ballance A.M. Suquet C. Hurst J.K. Pathways for intracellular generation of oxidants and tyrosine nitration by a macrophage cell line.Biochemistry. 2007; 46: 7536-7548Crossref PubMed Scopus (40) Google Scholar Various amounts (in micromoles per liter) of CM radical were measured to generate a standard curve to quantify the amount of oxidants produced by wild-type and EC-SOD KO cells. EPR settings were as follows: field sweep, 50 G; microwave frequency, 9.78 GHz; microwave power 20 mW, modulation amplitude, 2 G; conversion time, 327 ms; time constant, 655 ms; and receiver gain, 1 × 105. All buffers were treated with Chelex resin, and contained 25 μmol/L deferoxamine. Absence of transition metals was confirmed by the inability to detect the ascorbyl radical on exposure of buffer to 100 μmol/L ascorbic acid. Extracellular superoxide generation produced by PMA-stimulated peritoneal macrophages isolated from wild-type and EC-SOD KO mice was also measured via reduction of partially acetylated cytochrome c, which was calculated as the rate of change in absorbance at 550 nm over 2 minutes. Enhanced green fluorescent protein (EGFP)–expressing E. coli (BL21 strain)34Hu P.Q. Tuma-Warrino R.J. Bryan M.A. Mitchell K.G. Higgins D.E. Watkins S.C. Salter R.D. Escherichia coli expressing recombinant antigen and listeriolysin O stimulate class I–restricted CD8+ T cells following uptake by human APC.J Immunol. 2004; 172: 1595-1601PubMed Google Scholar, 35Salter R.D. Tuma-Warrino R.J. Hu P.Q. Watkins S.C. Rapid and extensive membrane reorganization by dendritic cells following exposure to bacteria revealed by high-resolution imaging.J Leukoc Biol. 2004; 75: 240-243Crossref PubMed Scopus (15) Google Scholar were grown overnight in Luria-Bertani broth with 50 μg/mL ampicillin at 37°C with continuous shaking. Cultures were refreshed and grown for 2 hours to reach log phase, washed with Dulbecco's PBS without calcium and magnesium, and used for phagocytosis and bacteria killing studies. Macrophages derived from bone marrow from wild-type and EC-SOD KO mice (500,000 cells per tube) were incubated in suspension with EGFP-expressing E. coli at 37°C (cell/bacteria, 1:100) in media lacking both antibiotics and L929 supplement.28Rhoades E.R. Orme I.M. Similar responses by macrophages from young and old mice infected with Mycobacterium tuberculosis.Mechanisms Ageing Dev. 1998; 106: 145-153Crossref PubMed Scopus (28) Google Scholar, 29Tse H.M. Josephy S.I. Chan E.D. Fouts D. Cooper A.M. Activation of the mitogen-activated protein kinase signaling pathway is instrumental in determining the ability of Mycobacterium avium to grow in murine macrophages.J Immunol. 2002; 168: 825-833PubMed Google Scholar Wild-type and EC-SOD KO cells and bacteria were incubated independently as controls. After 1 hour of incubation in a static tube, the bacteria remaining were cultured in Luria-Bertani agar using the pour plate method to determine the number of CFUs. The percentage of bacteria killed was calculated as the mean number of CFUs of the bacteria alone minus the CFUs remaining after 1 hour of incubation with cells, divided by the mean number of CFUs of the bacteria alone, multiplied by 100. Macrophages derived from bone marrow (1 × 105 cells per dish) were plated onto 35-mm collagen-coated glass-bottomed dishes (MatTek Corp, Ashland, MA). Multimode imaging (dimensions: X, Y, Z, time, and color) was used to image cellular interactions between phagocytes and bacteria in a temperature-controlled chamber (Tokai HIT Co., Ltd., Tokyo, Japan) at 37°C. Movies were obtained using a Nikon Ti inverted microscope (×40 magnification) running NIS-Elements AR 3.1 software (Nikon Instruments Inc., Melville, NY). Images were collected sequentially using a CoolSNAP HQ2 camera (Photometrics Ltd., Tucson, AZ) using shuttered illumination in both fluorescence and differential interference contrast. Movies were analyzed using NIS-Elements software. Phagocytosis was quantified over 1 hour by an observer (T.D.O.) blinded to the experimental groups. Macrophages derived from bone marrow (250,000 cells per tube) were incubated with 5 μg/mL Alexa Fluor 555 dextran (Molecular Probes) for 1 hour at either 4°C or 37°C. Cells were then fixed, and flow cytometric analysis was performed using a FACS Vantage along with DiVa and CellQuest analytical software (Becton Dickinson & Co., Franklin Lakes, NJ). To determine whether there were differences in cell viability between macrophages from wild-type and EC-SOD KO mice, bone marrow–derived macrophages were treated with various concentrations of PMA (Sigma-Aldridge Corp., St. Louis, MO), lipopolysaccharide (E. coli O26:B6; Sigma-Aldrich Corp.), and EGFP-expressing E. coli.34Hu P.Q. Tuma-Warrino R.J. Bryan M.A. Mitchell K.G. Higgins D.E. Watkins S.C. Salter R.D. Escherichia coli expressing recombinant antigen and listeriolysin O stimulate class I–restricted CD8+ T cells following uptake by human APC.J Immunol. 2004; 172: 1595-1601PubMed Google Scholar, 35Salter R.D. Tuma-Warrino R.J. Hu P.Q. Watkins S.C. Rapid and extensive membrane reorganization by dendritic cells following exposure to bacteria revealed by high-resolution imaging.J Leukoc Biol. 2004; 75: 240-243Crossref PubMed Scopus (15) Google Scholar Cell viability was measured using the CellTiter 96 AQueous Non-Radioactive Assay (Promega Corp., Madison, WI) according to the manufacturer's instructions or by means of visual assessment using trypan blue. Data were analyzed using commercially available software (PRISM version 5; GraphPad Software Inc., San Diego, CA). Animal experiments were analyzed using two-way analysis of variance with a Bonferroni posttest. Comparisons with one variable were analyzed using an unpaired Student's t-test. The number of cells that phagocytosed bacteria were compared using Fisher's exact test. Unless otherwise noted, all values are given as mean ± SEM. P < 0.05 was considered statistically significant. EC-SOD attenuates inflammation and oxidative injury in numerous pulmonary diseases; however, few studies have investigated its role in limiting injury in response to bacterial infections. To determine the role of EC-SOD in E. coli pneumonia, wild-type C57BL/6 and EC-SOD KO mice (congenic with the C57BL/6 strain) received an intratracheal instillation of E. coli and were euthanized immediately (0 hours) or at 6 or 24 hours after inoculation. The expression of EC-SOD in the airspace and lung parenchyma was evaluated in wild-type mice via Western blot analyses of the BALF and lung homogenates. Consistent with previous work,19Tan R.J. Lee J.S. Manni M.L. Fattman C.L. Tobolewski J.M. Zheng M. Kolls J.K. Martin T.R. Oury T.D. Inflammatory cells as a source of airspace extracellular superoxide dismutase after pulmonary injury.Am J Respir Cell Mol Biol. 2006; 34: 226-232Crossref PubMed Scopus (34) Google Scholar bacterial pneumonia leads to a significant increase in EC-SOD in the alveolar lining fluid (data not shown). To evaluate the importance of EC-SOD in response to bacterial infection, pulmonary inflammation was assessed in the BALF and lung tissue of infected wild-type and EC-SOD KO mice. There was no significant difference in inflammation between wild-type and EC-SOD KO animals at 6 hours after E. coli inoculation based on the cellular content of the BALF and the level of myeloperoxidase in the lung (data not shown). However, histologic examination of the lungs at 24 hours after inoculation with E. coli revealed an increased number of inflammatory cells in the EC-SOD KO mice compared with the wild-type mice (Figure 1A). EC-SOD KO mice also had more inflammatory cells in the BALF than did wild-type mice at 24 hours after infection (Figure 1B). Most cells in the lung and BALF were neutrophils, although macrophages were also present. At 24 hours after E. coli inoculation there were significantly greater numbers of neutrophils in the BALF from EC-SOD KO mice compared with wild-type mice (9.604 × 105 ± 1.625 versus 5.270 × 105 ± 0.635; Figure 1C) and higher myeloperoxidase activity in the lungs of EC-SOD KO mice compared with wild-type mice (Figure 1D). EC-SOD KO mice also exhibited significantly more protein in the BALF than did wild-type mice at 24 hours after infection (Figure 1E). These results are consistent with those of previous studies that demonstrated that EC-SOD expression in the lung inhibits inflammation in response to a wide variety of pulmonary injuries.11Fattman C.L. Tan R.J. Tobolewski J.M. Oury T.D. Increased sensitivity to asbestos-induced lung injury in mice lacking extracellular superoxide dismutase.Free Radic Biol Med. 2006; 40: 601-607Crossref PubMed Scopus (77) Google Scholar, 12Tan R.J. Fattman C.L. Watkins S.C. Oury T.D. Redistribution of pulmonary EC-SOD after exposure to asbestos.J Appl Physiol. 2004; 97: 2006-2013Crossref PubMed Scopus (68) Google Scholar, 13Fattman C.L. Chang L.Y. Termin T.A. Petersen L. Enghild J.J. Oury T.D. Enhanced bleomycin-induced pulmonary damage in mice lacking extracellular superoxide dismutase.Free Radic Biol Med. 2003; 35: 763-771Crossref PubMed Scopus (104) Google Scholar, 19Tan R.J. Lee J.S. Manni M.L. Fattman C.L. Tobolewski J.M. Zheng M. Kolls J.K. Martin T.R. Oury T.D. Inflammatory cells as a source of airspace extracellular superoxide dismutase after pulmonary injury.Am J Respir Cell Mol Biol. 2006; 34: 226-232Crossre

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