An investigation of IL-8 degradation in response to PA401 compared to hypertonic saline in bronchoalveolar lavage fluid of Cystic Fibrosis patients

The lung pathogenesis of cystic fibrosis (CF) involves inflammation, airway obstruction and an increased incidence of pulmonary infections. Increased levels of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) play a pivotal role in sustaining the cycle of inflammation in the CF lung. Glycosaminoglycans (GAGs) possess the ability to bind IL-8 providing protection from proteolytic degradation and maintaining it in an active state leading to sustained neutrophil chemotaxis. It has been shown that hypertonic saline (HTS) disrupts GAG:IL-8 complexes, thus rendering IL-8 susceptible to proteolysis thereby reducing neutrophil chemotaxis. The recombinant IL-8 decoy (PA401) binds glycans with higher affinity (x 40) than native IL-8. In this study, we compared the ability of PA401 and HTS to disrupt IL-8:GAG complexes in CF BALF.

IL-8 concentration in CF BALF was determined following exposure to PA401 or HTS by ELISA. PA401 degradation in CF BALF (± protease inhibitors) was examined using gradient SDS-PAGE and Western Blot analysis employing a primary antibody specific for the PA401 decoy (MAB8A12).

Individual CF BALF samples (n=7) displayed a high level of variability with regard to IL-8 concentration and response to PA401 or HTS. Exposure of pooled CF BAL to increasing concentrations of PA401 lead to a significant decrease in the level of detectable IL-8 (p<0.05) and neutrophil chemotactic efficiency (30 %, p<0.05). Significantly reduced levels of IL-8 (p<0.05) were detected following incubation with PA401 for 4 hr in 6/7 individuals with CF when compared to a PBS control. The level of IL-8 present in BALF following incubation with PA401 was significantly reduced compared to HTS (p<0.05) in 2/3 CF patients. Western Blot analysis indicated that serine proteases (inhibited by alpha-1 antitrypsin, PMSF and pefabloc) play a major role in degrading PA401.

The reduced levels of IL-8 in BALF samples treated with PA401 revealed that PA401 is likely to be effective in disrupting IL-8:GAG complexes in the CF lung rendering IL-8 susceptible to proteolysis and thus may be seen as a therapeutic target in the treatment of CF. Further benefits of PA401 are evident as the decoy did not accumulate in CF samples and post IL-8 clearance, it too was degraded by serine and metalloproteases. Clinical application of an IL-8 decoy may serve to decrease the inflammatory burden in the CF lung in vivo.