Modulation of neutrophil NADPH oxidase activity by alpha-1 antitrypsin

Alpha-1 antitrypsin is a serine protease inhibitor that demonstrates an array of additional immunomodulatory functions. Individuals with the genetic condition, alpha-1 antitrypsin deficiency (AATD) are at increased risk of early onset emphysematous lung disease. This lung disease is partly driven by neutrophil mediated lung destruction in an environment of low AAT. The diverse functions of AAT appear to point to the pathogenesis of lung disease in AATD being more complex than a mere protease/anti-protease imbalance. There is growing evidence of the role of immune cell derived oxidative stress in the pathogenesis of non-genetic COPD. No such role has been firmly established in AATD related lung disease, while there is some evidence that AAT may affect immune cell O₂^- production, based on in vitro studies. The aim of this study was to examine the effects of AAT on neutrophil reactive oxygen species (ROS) production and to determine ROS production by circulating neutrophils in AATD individuals.

We investigated the effects of AAT on circulating neutrophils that had been exposed to fMLP and IL-8 using a cytochrome c reduction assay. We found that physiological concentrations of AAT were capable of significantly modulating neutrophil O₂^- production. We confirmed that the effects of AAT were directly related to NADPH oxidase activity by measuring O₂ consumption in these cells and by demonstrating the assembly of NADPH oxidase subunits at the neutrophil cell membrane. Further to this, we showed that circulating neutrophils from AATD individuals exhibit enhanced O₂^- production in response to soluble stimuli (fMLP). This enhanced activity did not affect the cytosolic pH of the neutrophil, despite our finding of a reduced abundance of HVCN1 in AATD neutrophils. The mechanism of enhanced responsiveness to fMLP by AATD neutrophils appears to be related to the ability of AAT to bind fMLP and prevent engagement with FPR1 on the cell membrane. AAT augmentation therapy was shown to reduce neutrophil O₂^- production. In summary, this study demonstrates that AAT can bind fMLP, preventing engagement with FPR1. This interaction prevents the assembly of NADPH oxidase at the cell membrane and therefore reduces ROS production. In AATD individuals this equates to a more robust ROS production in response to stimuli, an abnormality which is corrected by the administration of AAT augmentation therapy.