Effect of Nitrogen Dioxide on Synthesis of Inflammatory Cytokines Expressed by Human Bronchial Epithelial Cells In Vitro

Abstract

Abstract Although studies of nitrogen dioxide (NO2) inhalation, in both animals and humans, have demonstrated that this agent can cause epithelial cell damage and inflammation of the airway epithelium, the mechanisms underlying these effects are not well understood. We have cultured human bronchial epithelial cells, as explant cultures from surgical tissue, and studied these firstly from their ability to constitutively synthesize specific proinflammatory cytokines and then investigated the effect of exposure to NO2 on the generation of these cytokines. Constitutive synthesis of cytokines was evaluated by analysis of both the expression of the mRNA for interleukin (IL)-1β, IL-4, IL-8, granulocyte/macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), by the polymerase chain reaction (PCR), and by immunocytochemical staining for the presence of cell-associated IL-1β, IL-8, GM-CSF, TNF-α, and IFN-γ, using specific monoclonal and polyclonal antibodies directed towards these cytokines. Release of IL-4, IL-8, GM-CSF, TNF-α, and IFN-γ following exposure to 5% CO2 in air or 400 ppb and 800 ppb NO2 for 6 h was investigated by enzyme-linked immunosorbent assay. PCR demonstrated that the human bronchial epithelial cells expressed the mRNA for IL-1β, IL-8, GM-CSF, and TNF-α but not for IL-4 and IFN-γ. Immunocytochemical staining confirmed the presence of endogenous IL-1β, IL-8, GM-CSF, and TNF-α. Analysis of cultures for cytokines released into the culture medium demonstrated that the untreated control cells released 0.13 ± 0.05 pg GM-CSF µg−1 cellular protein, 11.18 ± 1.71 pg IL-8 µg−1 cellular protein, and 0.06 ± 0.02 pg TNF-αµg− cellular protein after 6 h of exposure to 5% CO2 in air. Exposure of cultures to 400 ppb NO2 for 6 h, however, significantly increased the release of GM-CSF (0.44 ± 0.10 pg GM-CSF µg−1 cellular protein, P < 0.01), IL-8 (22.32 ± 3.65 pg IL-8 µg−1 cellular protein, P < 0.01), and TNF-α (2.93 ± 0.51 pg TNF-αµg−1 cellular protein, P < 0.001). In contrast, exposure of cultures to 800 ppb NO2 led to significant release of only GM-CSF (0.29 ± 0.06 pg GM-CSF µg−1 cellular protein, P < 0.05) after 6 h of exposure. Release of neither IL-4 nor IFN-γ was detected in any control cultures or cultures exposed to 400 ppb or 800 ppb NO2 under these incubation conditions. These studies suggest that exposure to NO2, at concentrations found at the curbside in heavy traffic conditions or indoors in households with gas cooking stoves, may induce the synthesis of proinflammatory cytokines from airway epithelial cells and consequently play an important role in the etiology of airway disease.