Manzo, N; Richards, J; Slade, R; Lagier, A; Dye, J
RATIONALE: Individuals with chronic pulmonary inflammation due to disease are more susceptible to the adverse health effects associated with exposure to particulate matter (PM) air pollutants, such as diesel exhaust particles (DEP). Increasing evidence suggests that these adverse effects are in part mediated by reactive oxygen species (ROS), such as superoxide (O2-). However, individuals with pulmonary inflammation also have increased levels of nitric oxide (NO), which can react with O2- to produce more potent radicals with damaging cellular effects. Using an analogous in vitro system that models “healthy” and “inflamed” (susceptible) epithelial cells by cytokine treatment, we have previously shown that DEP exposure of cytokine-treated epithelial cells results in oxidative stress and cell injury. The goal of the current study was to further investigate the role of O2- and NO in this differential response.
METHODS: LA-4 cells, a murine lung epithelial cell line, were treated with pro-inflammatory cytokines (cytomix: TNFα+IL-1β+IFNγ, 0.2ng/mL, 24hr) to produce an inflammatory microenvironment. Inducible nitric oxide synthase (iNOS) mRNA and protein levels were evaluated in control and cytomix-treated cells, with or without an iNOS specific inhibitor (1400W; 10μM). Similarly, NO production from control and cytomix-treated cells was quantified using the fluorescent probe DAF-2 diacetate and the griess reaction. ROS production, including O2-, in control cells exposed to DEP (25μg/cm2, 2hr) was determined using the fluorescent probes H2DCFDA and dihydroethidium (DHE), with or without superoxide dismutase (SOD; 200U/mL). Lastly, ROS production from control and cytomix-treated cells exposed to DEP was quantified by H2DCFDA with or without SOD and 1400W treatment.
RESULTS: Whereas control cells released little NO, cytomix treatment increased NO production, via increased iNOS mRNA expression and protein levels, which was inhibited by 1400W. Exposure of control cells to DEP resulted in increased ROS that was inhibited by SOD. Furthermore, DEP exposure of control cells produced O2-, evident by increased nuclear and cytoplasmic fluorescence of DHE. DEP exposure of cytomix-treated cells resulted in even greater ROS production than control cells exposed to DEP, which was significantly reduced by treatment with 1400W and SOD.
CONCLUSIONS: The exposure of cytomix-treated LA-4 epithelial cells to DEP resulted in increased production of ROS through the combined effects of NO and O2-, culminating in oxidative stress and overt cell injury. Similar mechanisms may account for the increased susceptibility of individuals with pre-existing inflammatory lung diseases, like asthma and COPD, and may exacerbate lung injury caused by DEP exposure.
