ABSTRACT
Extracellular superoxide dismutase (EC-SOD) is an abundant antioxidant in the lung and vascular walls. Previous studies have shown that EC-SOD attenuates lung injury in a diverse variety of lung injury models. In this study, we examined the role of EC-SOD in mediating lipopolysaccharide (LPS)-induced lung inflammation. We found that LPS-induced neutrophilic lung inflammation was exaggerated in EC-SOD-deficient mice and diminished in mice that overexpressed EC-SOD specifically in the lung. Similar patterns were seen for bronchoalveolar lavage cytokines, such as tumor necrosis factor-alpha, keratinocyte-derived chemokines, and macrophage inflammatory protein-2 as well as expression of lung intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial cell selectin, and platelet selectin. In a macrophage cell line, EC-SOD inhibited LPS-induced macrophage cytokine release, but did not alter expression of intercellular adhesion molecules in endothelial cells. These results suggest that EC-SOD plays an important role in attenuating the inflammatory response in the lung most likely by decreasing release of proinflammatory cytokines from phagocytes.
Subject(s)
Cell Adhesion , Extracellular Fluid/enzymology , Lipopolysaccharides/pharmacology , Lung/physiopathology , Pneumonia/etiology , Superoxide Dismutase/pharmacology , Animals , Biomarkers/analysis , Bronchoalveolar Lavage Fluid , Female , Gene Expression Regulation, Enzymologic , Humans , Intercellular Adhesion Molecule-1/metabolism , Lung/enzymology , Macrophages/cytology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neutrophils/drug effects , Neutrophils/enzymology , Neutrophils/immunology , Neutrophils/metabolism , Peroxidase/metabolism , Phagocytes/cytology , Phagocytes/metabolism , Pneumonia/enzymology , Pneumonia/physiopathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Selectins/metabolism , Superoxide Dismutase/metabolism , Tumor Necrosis Factor-alpha/metabolism , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
The usual methods for preparing lungs for morphologic study involve the instillation of fixatives that modifyproteins and RNA such that the tissue is unsuitable for molecular studies. To develop a technique suitable for molecular studies, pieces of adult rat lungs were infiltrated with agarose, glutaraldehyde, or paraformaldehyde and the consistency of alveolar inflation was compared to lungs inflated with 10% formalin. Only direct injection with 1% agarose resulted in comparable inflation of lung tissue and preserved RNA and protein. Thus, this technique enables simultaneous molecular and morphometric analysis of the lung on small pieces of lung tissue in heterogeneous lung diseases.
Subject(s)
Lung , Organ Preservation Solutions , Organ Preservation/methods , Sepharose , Tissue Fixation/methods , Animals , Formaldehyde , Glutaral , Image Processing, Computer-Assisted , Immunohistochemistry , Injections , Male , Polymers , Proteins/analysis , Pulmonary Alveoli/anatomy & histology , Pulmonary Alveoli/chemistry , RNA/analysis , Rats , Rats, Sprague-DawleyABSTRACT
Bleomycin administration results in well-described intracellular oxidative stress that can lead to pulmonary fibrosis. The role of alveolar interstitial antioxidants in this model is unknown. Extracellular superoxide dismutase (EC-SOD) is the primary endogenous extracellular antioxidant enzyme and is abundant in the lung. We hypothesized that EC-SOD plays an important role in attenuating bleomycin-induced lung injury. Two weeks after intratracheal bleomycin administration, we found that wild-type mice induced a 106 +/- 25% increase in lung EC-SOD. Immunohistochemical staining revealed that a large increase in EC-SOD occurred in injured lung. Using mice that overexpress EC-SOD specifically in the lung, we found a 53 +/- 14% reduction in bleomycin-induced lung injury assessed histologically and a 17 +/- 6% reduction in lung collagen content 2 wk after bleomycin administration. We conclude that EC-SOD plays an important role in reducing the magnitude of lung injury from extracellular free radicals after bleomycin administration.
Subject(s)
Pulmonary Fibrosis/enzymology , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Animals , Antibiotics, Antineoplastic , Bleomycin , Collagen/metabolism , Extracellular Space/enzymology , Immunohistochemistry , Lung/enzymology , Lung/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oxidation-Reduction , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/pathology , Superoxide Dismutase/analysisABSTRACT
Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SOD's tissue half-life and affinity for heparin. There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D. (1999) J. Biol. Chem. 274, 14818-14822). By using mammalian cell lines, we have now determined that removal of the heparin-binding region occurs after passage through the Golgi network but before being secreted into the extracellular space. Specific protease inhibitors and overexpression of intracellular proteases implicate furin as a processing protease. In vitro experiments using furin and purified EC-SOD suggest that furin proteolytically cleaves EC-SOD in the middle of the polybasic region and then requires an additional carboxypeptidase to remove the remaining lysines and arginines. A mutation in Arg(213) renders EC-SOD resistant to furin processing. These results indicate that furin-dependent processing of EC-SOD is important for determining the tissue distribution and half-life of EC-SOD.
