Molecular mechanisms of nitrogen dioxide induced lung damage
Author: Tu, Ba
Date: 1995-12-08
Location: Seminarierummet, Rättsmedicinska institutionens föreläsningssal (Doktorsringen 16A) Karolinska institutet
Time: 9.00
Department: Institutet för miljömedicin / Institute of Environmental Medicine
Abstract
Nitrogen dioxide (NO2) is an oxidant gas which contaminates ambient air in many urban and industrial locations, and indoor air in homes with combustion appliances. The Environmental Protection Agency (USA) regulates N02 in ambient air as a "criteria" pollutant. In spite of decades of laboratory, clinical, and epidemiological research, the mechanisms of the well-known adverse effects of NO2 exposure on the lung, the most important target for this gas, are not well understood. Of particular interest are the molecular mechanisms behind the airways inflammation induced by this gas which are far from clear. The general aims of this study: 1. To establish cell culture models and an in vitro gas exposure model for mechanistic studies of factors underlying the pulmonary toxicity of the NO2 and, eventually, 03. 2. To define the cytoprotective roles of major airway antioxidants in the alveolar structure against NO2. 3.
To study the molecular mechanisms of NO2-induced airway inflammation.We developed a new, optimal in vitro gas exposure modd for the exposure of inverted cell cultures to NO2, which present several advantages. Firstly, the cell monolayer can be directly exposed toNO2 in the gas phase for up to 1 hour, without the interposition of a thick aqueous layer. Secondly, the chamber system allows the simple and precise control of the gas concentration during the exposure. Finally, the system allows the simultaneous exposure of large numbers of cells under sterile conditions, facilitating further culture of the cells after the exposure. By using this gas exposure model, the relative importance of water-soluble nonenzymatic antioxidant components in airway epithelial lining fluids, ascorbate and glutathione (GSH), in protecting human cells against NO2-induced cytotoxicity has been evaluated. From the results we conclude that both ascorbate and GSH play important roles in defending human cells from the toxicity of NO2 under direct exposure conditions and that both extracellular and intracellular ascorbate can very efficiently play a protective role. The results also strengthen the premise that ascorbate and GSH co-operate in such antioxidative protection. Chemical oxidation assays illustrated that NO2 directly reacts with ascorbate and GSH being itself reduced to nitrite. Considering alveolar macrophages (AMs) may be direct cellular target of NO2 in vivo and considering their important potential roles of the cells in airway inflammation due to their capacity of releasing many kind of inflammatory mediators including pro inflammatory cytokines and chemokines, a murine macrophage cell line IC-21 and human primary alveolar macrophages were directly exposed to NO2 using our modd.
Results indicated that NO2 does not stimulate the mRNA expression and protein release of TNF-ac, IL-l~, IL,8 and MIP-l from these cells. Our knowledge of understanding the involvement of endothelial and epithelial cell metabolism in airway inflammation, due partially to their capacity to produce and release proinflammatory cytokines and chemokines, has been increasing rapidly. Toxicological data has indicated that airway epithelial and alveolar endothelial cells may be damaged directly and/or indirectly by inhaled N02. Therefore, we further studied the effects of NO2 on expression of proinflammatory cytokines, chemokines and infammatory cell adhesion molecules in human endothelial cells, airway epithelial cells and bronchial biopsies. The results from RT-PCR, semi-quantitative RT-PCR and ELISA show that NO2 does not induce or stimulate the expression of inflammatory cytokines, chemokines or cell adhesion molecules in any of the probed cells/tissues under the respective condition of exposure. In summary, the in vitro gaseous exposure model is a suitable system for the study of, the biological effects of oxidative gaseous pollutants. Both ascorbate and GSH dependently and independently protect human cells against the NO2-induced cytotoxicity. More sensitive tools and more quantitative studies are required for understanding the involvement of proinflammatory cytokines, chemokines and inflammatory cell adhesion molecules as well as other mediator, from different cell types in airways, in NO2-induced airways inflammation.
To study the molecular mechanisms of NO2-induced airway inflammation.We developed a new, optimal in vitro gas exposure modd for the exposure of inverted cell cultures to NO2, which present several advantages. Firstly, the cell monolayer can be directly exposed toNO2 in the gas phase for up to 1 hour, without the interposition of a thick aqueous layer. Secondly, the chamber system allows the simple and precise control of the gas concentration during the exposure. Finally, the system allows the simultaneous exposure of large numbers of cells under sterile conditions, facilitating further culture of the cells after the exposure. By using this gas exposure model, the relative importance of water-soluble nonenzymatic antioxidant components in airway epithelial lining fluids, ascorbate and glutathione (GSH), in protecting human cells against NO2-induced cytotoxicity has been evaluated. From the results we conclude that both ascorbate and GSH play important roles in defending human cells from the toxicity of NO2 under direct exposure conditions and that both extracellular and intracellular ascorbate can very efficiently play a protective role. The results also strengthen the premise that ascorbate and GSH co-operate in such antioxidative protection. Chemical oxidation assays illustrated that NO2 directly reacts with ascorbate and GSH being itself reduced to nitrite. Considering alveolar macrophages (AMs) may be direct cellular target of NO2 in vivo and considering their important potential roles of the cells in airway inflammation due to their capacity of releasing many kind of inflammatory mediators including pro inflammatory cytokines and chemokines, a murine macrophage cell line IC-21 and human primary alveolar macrophages were directly exposed to NO2 using our modd.
Results indicated that NO2 does not stimulate the mRNA expression and protein release of TNF-ac, IL-l~, IL,8 and MIP-l from these cells. Our knowledge of understanding the involvement of endothelial and epithelial cell metabolism in airway inflammation, due partially to their capacity to produce and release proinflammatory cytokines and chemokines, has been increasing rapidly. Toxicological data has indicated that airway epithelial and alveolar endothelial cells may be damaged directly and/or indirectly by inhaled N02. Therefore, we further studied the effects of NO2 on expression of proinflammatory cytokines, chemokines and infammatory cell adhesion molecules in human endothelial cells, airway epithelial cells and bronchial biopsies. The results from RT-PCR, semi-quantitative RT-PCR and ELISA show that NO2 does not induce or stimulate the expression of inflammatory cytokines, chemokines or cell adhesion molecules in any of the probed cells/tissues under the respective condition of exposure. In summary, the in vitro gaseous exposure model is a suitable system for the study of, the biological effects of oxidative gaseous pollutants. Both ascorbate and GSH dependently and independently protect human cells against the NO2-induced cytotoxicity. More sensitive tools and more quantitative studies are required for understanding the involvement of proinflammatory cytokines, chemokines and inflammatory cell adhesion molecules as well as other mediator, from different cell types in airways, in NO2-induced airways inflammation.
Issue date: 1995-11-17
Publication year: 1995
ISBN: 91-628-1837-6
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