PURIFICATION AND CHARACTERIZATION OF DEFENSINS FROM CYSTIC-FIBROSIS SPUTUM

Objective and Design: Cystic fibrosis (CF) sputum contains large numbe rs 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 possib ility that defensins damage CF airways, this study examines the concen tration and properties of defensins in CF sputum. Materials: As a sour ce of biological material in which to assay levels of defensins, purul ent sputum was collected from persons with CF hospitalized with exacer bations of bronchitis. Purulent CF sputum was also a source of materia l for purification of defensins. Methods: Defensin concentration in th e cell-free component of CF sputum was measured by immunoassay. Defens ins acid-extracted from sputum were purified by Sephacryl S-200 gel fi ltration chromatography and reverse phase-high pressure liquid chromat ography (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 ra nged from 300 to > 1600 mu g/ml, higher than levels previously reporte d 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. Conclusion s: This study suggests that defensins in CF sputum are intact and suff iciently abundant that they may damage airway epithelium.