Cell-free supernatant of Pseudomonas aeruginosa (PA) recruits neutrophils into the airways indirectly by inducing the production of chemotactic factors, including interleukin-8 (IL-8). PA products stimulate IL-8 expression selectively in surface airway epithelium, gland ducts, serous cells, and recruited neutrophils. To examine the relative contribution of neutrophils in IL-8 release in the airway lumen, we studied the effect of inhibition of neutrophil recruitment on IL-8 concentration in tracheal fluid after introduction of PA supernatant into the dog trachea in vivo. Tracheal superfusion with PA supernatant caused neutrophil recruitment and increased the IL-8 concentration in the tracheal lumen; NPC 15669 inhibited both effects. To study whether migration of neutrophils into the airway lumen per se induces their expression of IL-8, we compared effects of local introduction of IL-8 and of PA supernatant into the trachea on IL-8 expression in neutrophils recruited into the trachea. PA supernatant, but not exogenous IL-8 alone, induced IL-8 mRNA expression in neutrophils recruited into the trachea. To determine what product(s) of PA stimulate IL-8 expression in neutrophils, we examined neutrophils isolated from peripheral blood. PA supernatant induced IL-8 production in neutrophils, an effect reproduced by PA lipopolysaccharide and inhibited by polymyxin B. These results suggest that neutrophils recruited into the airway lumen play a major role in local IL-8 production in airways in response to bacteria such as PA, depending on the presence of stimuli such as lipopolysaccharide.