Purification and characterization of defensins from cystic fibrosis sputum

Objective and Design: Cystic fibrosis (CF) sputum contains large numbers of neutrophils whose most abundant granule proteins are defensins. Within phagolysosomes, defensins kill microbes; however, extracellular defensins can be toxic to human cells. To begin to explore the possibility that defensins damage CF airways, this study examines the concentration and properties of defensins in CF sputum. Materials: As a source of biological material in which to assay levels of defensins, purulent sputum was collected from persons with CF hospitalized with exacerbations of bronchitis. Purulent CF sputum was also a source of material for purification of defensins. Methods: Defensin concentration in the cell-free component of CF sputum was measured by immunoassay. Defensins acid-extracted from sputum were purified by Sephacryl S-200 gel filtration chromatography and reverse phase-high pressure liquid chromatography (HPLC). Toxicity of CF defensins was tested by incubating the purified defensins with a line of CF tracheal cells cultured in serum-free medium. Results: In 5 patients with CF, sputum defensin levels ranged from 300 to > 1600 mu g/ml, higher than levels previously reported in any human secretion or fluid and greatly exceeding concentrations toxic to mammalian cells in vitro. HPCL-purified CF sputum defensins were pure as judged by acid urea-PAGE and N-terminal sequencing, which revealed a mixture of defensins-1, -2 and -3 at ratios of similar to 4:2:1. At > 10 mu g/ml the purified mixture was toxic for a line of CF tracheal cells cultured in serum-free medium, as judged by reductions in cell numbers and increased permeability to trypan blue. Conclusions: This study suggests that defensins in CF sputum are intact and sufficiently abundant that they may damage airway epithelium.