Cigarette Smoke Stimulates Release of Neutrophil Chemotactic Activity from Cultured Bovine Bronchial Epithelial Cells

Abstract

1. Recruitment of neutrophils into the airway is a prominent feature of chronic bronchitis, a syndrome often associated with exposure to cigarette smoke. Since bronchial epithelial cells are the ‘first’ lung cells to come into contact with smoke, these cells may be responsible for neutrophil recruitment into the airway by release of neutrophil chemotactic activity in response to cigarette smoke. 2. To evaluate this hypothesis, we prepared bovine bronchial epithelial cells and measured their ability to release neutrophil chemotactic activity following exposure to cigarette smoke. Bronchial epithelial cells were prepared by overnight digestion with protease, filtered through 100-μm Nitex mesh and resuspended in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and antibiotics and cultured at 2×106 cells in 2 ml of medium in 35-mm culture dishes. After 4 days, dishes were rinsed and refed with medium without fetal calf serum and incubated with and without 1:10 diluted smoke extract for 6 h at 37°C. The neutrophil chemotactic activity of the supernatant fluids was measured by the blindwell chamber technique. 3. Cigarette smoke itself added to medium did not stimulate chemotaxis. In contrast, cigarette smoke did stimulate the release of neutrophil chemotactic activity from bovine bronchial epithelial cells [15 ± 3 (control) versus 74 ± 5 (smoke), P < 0.01]. 4. This neutrophil chemotactic activity was dialysable, pepsin and acid stable, heat sensitive and lipid extractable. Sephadex G-75 column chromatography demonstrated two peaks of neutrophil chemotactic activity. 5. The lipoxygenase inhibitors diethylcarbamazine and nordihydroguaratic acid both diminished the release of chemotactic activity, suggesting that the activity may be a lipoxygenase product(s). 6. Dibutyryl cyclic AMP also blocked the smoke-stimulated release of neutrophil chemotactic activity, suggesting that its release may be regulated by intracellular cyclic AMP. 7. Thus, bovine bronchial epithelial cells release neutrophil chemotactic activity in response to cigarette smoke, suggesting that bronchial epithelial cells may be modulators of the airway inflammatory response caused by cigarette smoke.