Date of Award

11-2016

Document type

Thesis

Degree Name

MD (Medical Doctor)

First Supervisor

Professor Noel G. McElvaney

Second Supervisor

Dr. Emer Reeves

Keywords

Alpha-1 Antitrypsin Deficiency, Neutrophil, Dysregulated Degranulation

Abstract

Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterised by excessive neutrophil degranulation and a protease: anti-protease imbalance leading to premature emphysema. Current specialised treatment for AATD consists of once weekly infusion of plasma purified AAT. Neutrophil degranulation is under the control of small GTP-binding proteins, including Ras-related C3 botulinum toxin substrate 2 (Rac2). The molecular basis for aberrant neutrophil degranulation in AATD has not been elucidated to date.

The aim of this study was to fully characterise neutrophil degranulation in AATD and to determine the effects of AAT augmentation therapy on the AATD neutrophil.

In this study, we examined degranulation by AATD neutrophils by Western blotting. This revealed a 3-fold increase in levels of myeloperoxidase (MPO), human cathelicidin antimicrobial protein (hCAP-18) and matrix metalloprotease-9 (MMP-9), markers of primary, secondary and tertiary granules, respectively (p=0.023, p=0.036 and p=0.042, respectively).

Excessive active Rac2 content of 239% was confirmed in AATD neutrophils using a Rac2 activation assay (p=0.016). In addition, fluorescence resonance energy transfer (FRET) and a cytochrome c reduction assay demonstrated elevated membrane-bound neutrophil elastase (NE) on AATD neutrophils (p=0.034) and a 107% increase in superoxide (O2-) production by NE-stimulated neutrophils (p=0.011), respectively. Furthermore, we have identified a novel pathway of neutrophil reactive oxygen species (ROS) production via activation of trans-membrane protease activated receptor 2 (PAR2) by NE and we have demonstrated the ability of exogenous AAT to correct O2- release by NE-stimulated neutrophils in-vitro (63.3% reduction, p=0.008).

Most importantly, this study confirmed for the first time that AAT augmentation therapy not only normalised excessive degranulation patterns but also resulted in a 75% decrease in elevated Rac2 activity by AATD neutrophils assessed by Western blotting ex-vivo (p=0.03).

In conclusion, this study demonstrated the ability of AAT to regulate aberrant degranulation by AATD neutrophils, supporting our hypothesis of the ability of AAT to restore protease: anti-protease balance and highlighted a further dimension to the benefits of AAT augmentation therapy for our AATD patients.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

5 MB

Comments

A thesis submitted for the degree of Doctor of Medicine from the Royal College of Surgeons in Ireland in 2016.

Available for download on Saturday, November 17, 2018

Share

COinS