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.
First SupervisorGerry McElvaney
Second SupervisorEmer Reeves
CommentsA thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2019.
Published CitationHawkins P. Modulation of neutrophil NADPH oxidase activity by alpha-1 antitrypsin [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2019.
Degree NameDoctor of Philosophy (PhD)
Date of award2019-11-30
- Doctor of Philosophy (PhD)