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A7423 - The Circulating Neutrophil Proteome Is Altered in Alpha-1 Antitrypsin Deficiency and Is Modified by Intravenous Augmentation Therapy
Author Block: T. McEnery1, O. Coleman2, P. Hawkins1, M. White1, N. Lacey1, P. Meleady2, E. P. Reeves1, N. G. McElvaney1; 1Respiratory Research, Royal College of Surgeons in Ireland, Dublin, Ireland, 2National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.
Introduction
Alpha-1 antitrypsin (AAT) deficiency (AATD) is the 3rd most common lethal genetic condition among Caucasians. AATD may result in early onset emphysema and liver disease. Treatment is available with weekly intravenous infusions of pooled human AAT (hAAT augmentation), which has been shown to slow progression of emphysema.
Lung disease in AATD is commonly thought to result from protease-antiprotease imbalance leading to tissue destruction and emphysema. Apart from its role as a serine protease inhibitor, increasing evidence points to the key anti-inflammatory effects of AAT. Our aim is to present the first proteomic analysis of the plasma membrane of circulating neutrophils in AATD, in order to further elucidate the important effects of AAT on the immune cell.
Methods
Neutrophils were isolated from ZZ-AATD patients on day 0 and day 2 of hAAT augmentation therapy (nadir and peak AAT levels respectively, n=6 per group), FEV1-matched COPD patients without AATD, ZZ-AATD patients not on augmentation therapy and healthy controls (n=12 per group). A plasma membrane fraction was prepared by sucrose density ultracentrifugation. Proteomic analysis was performed using liquid chromatography mass spectrometry following digestion by trypsin. Proteins were identified if ≥ 2 unique peptides were detected by MS. Differential expression was defined as a fold change in abundance of ≥1.5 with a p value of ≤ 0.05. Differences in protein expression were confirmed by Western blotting. Student’s t-test used to determine significance.
Results
A total of 1136 proteins were identified in the neutrophil plasma membrane. 228 of these were either down-regulated or absent on day 2 of hAAT augmentation compared to day 0. These included proteins representing neutrophil degranulation (proteinase 3 [p=0.03], lacto-transferrin [p=0.05], matrix metalloproteinase-9 [p=0.04]), cytoskeletal proteins (profilin [p= 0.04], coronin [p=0.03], alpha-actinin [p=0.009], filamin [p=0.03], tubulin and spectrin [p=0.04]) as well as proteins involved in cell adhesion (Integrin alpha-L, [p=0.03]) and apoptosis (annexin A1 [p=0.03]). Expression of CD16b was reduced in ZZ-AATD compared to healthy controls. Western blotting confirmed these results in ZZ-AATD patient samples compared to healthy control neutrophil membranes.
Discussion
We have shown that the proteome of the circulating neutrophil is altered in AATD and is modified by hAAT augmentation. Down-regulation of neutrophil granule and cytoskeletal proteins following hAAT augmentation suggest that this therapy reduces degranulation and priming of circulating neutrophils respectively. Our results indicate that altered neutrophil physiology is a key feature of AATD and that hAAT therapy modulates neutrophil priming, chemotaxis, adhesion, degranulation and apoptosis.