EFFECT OF NITROGEN-DIOXIDE ON SYNTHESIS OF INFLAMMATORY CYTOKINES EXPRESSED BY HUMAN BRONCHIAL EPITHELIAL-CELLS IN-VITRO

Although studies of nitrogen dioxide (NO2) inhalation, in both animals and humans, have demonstrated that this agent can cause epithelial ce ll damage and inflammation of the airway epithelium, the mechanisms un derlying 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 synthe size specific proinflammatory cytokines and then investigated the effe ct of exposure to NO2 on the generation of these cytokines. Constituti ve synthesis of cytokines was evaluated by analysis of both the expres sion of the mRNA for interleukin (IL)-1beta, IL-4, IL-8, granulocyte/m acrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-al pha (TNF-alpha), and interferon-gamma (IFN-gamma), by the polymerase c hain reaction (PCR), and by immunocytochemical staining for the presen ce of cell-associated IL-1beta, IL-8, GM-CSF, TNF-alpha, and IFN-gamma , using specific monoclonal and polyclonal antibodies directed towards these cytokines. Release of IL-4, IL-8, GM-CSF, TNF-alpha, and IFN-ga mma following exposure to 5% CO, in air or 400 ppb and 800 ppb NO, for 6 h was investigated by enzyme-linked immunosorbent assay. PCR demons trated that the human bronchial epithelial cells expressed the mRNA fo r IL-1beta, IL-8, GM-CSF, and TNF-alpha but not for IL-4 and IFN-gamma . Immunocytochemical staining confirmed the presence of endogenous IL- 1beta, IL-8, GM-CSF, and TNF-alpha. Analysis of cultures for cytokines released into the culture medium demonstrated that the untreated cont rol cells released 0.13 +/- 0.05 pg GM-CSF mug-1 cellular protein, 11. 18 +/- 1.71 pg IL-8 /mug-1 cellular protein, and 0.06 +/- 0.02 pg TNF- alpha mug-1 cellular protein after 6 h of exposure to 5% CO2 in air. E xposure of cultures to 400 ppb NO2 for 6 h, however, significantly inc reased the release of GM-CSF (0.44 +/- 0.10 pg GM-CSF mug-1 cellular p rotein, P < 0.01), IL-8 (22.32 +/- 3.65 pg IL-8 mug-1 cellular protein , P < 0.01), and TNF-alpha (2.93 +/- 0.51 pg TNF-alpha mug-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 mug -1 cellular protein, P < 0.05) after 6 h of exposure. Release of neith er IL-4 nor IFN-gamma was detected in any control cultures or cultures exposed to 400 ppb or 800 ppb NO, 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 wit h gas cooking stoves, may induce the synthesis of proinflammatory cyto kines from airway epithelial cells and consequently play an important role in the etiology of airway disease.