Neutrophil elastase and elastase-rich cystic fibrosis sputum degranulate human eosinophils in vitro

Neutrophils, eosinophils, and their proinflammatory constituents are import ant mediators of airway disease, and high levels of neutrophil proteases an d eosinophil cationic protein (ECP) are found in sputum from patients with cystic fibrosis (CF). To investigate whether neutrophil proteases or CF spu tum causes eosinophil degranulation, purified eosinophils from atopic asthm atic subjects were incubated for 2 h with neutrophil elastase, cathepsin G, and CF sputum, and the release of ECP was measured. We found that the perc ent release of ECP was higher after incubation with neutrophil elastase (10 (-5) M) than with a buffer control [6.1 +/- 0.8 (SE) vs. 1.7 +/- 0.1%; P < 0.003] and represented >50% of the release caused by positive controls [Ca2 + ionophore A-23187 (5 x 10(-6) M) or serum-coated Sephadex beads]. The rel ease of ECP after incubation with cathepsin G (2.3 +/- 0.2%) and CF sputum (6.2 +/- 2.0%) was also significantly higher than that with a buffer contro l (P < 0.05). Neutralization of free elastase activity with al-proteinase i nhibitor reduced the mean percent degranulation of eosinophils by neutrophi l elastase by 50% (P = 0.0004) and by CF sputum by 75% (P = 0.02). Preincub ation of eosinophils with cytochalasin B (10 mg/ml) and depletion of the in cubation medium of Ca2+ also significantly attenuated degranulation of eosi nophils incubated with purified free neutrophil elastase or CF sputum (P < 0.05). We conclude that neutrophil proteases, especially neutrophil elastas e, and elastase-rich CF sputum cause degranulation of eosinophils in a mech anism partially dependent on Ca2+ and actin filaments.