FINAL ID: 103
CONTACT NAME: Ciara O' Dwyer
CATEGORY: AIRWAYS PHYSIOLOGY, PATHOPHYSIOLOGY & DEFENSE
KEYWORDS: aerosol drug delivery, airway inflammation, Anti-inflammatory, leukotrienes, neutrophil
Abstract
TITLE: AEROSOLIZED ALPHA-1 ANTITRYPSIN AS A POSSIBLE THERAPY TARGETING LEUKOTRIENE B4 INDUCED INFLAMMATION IN CYSTIC
FIBROSIS
AUTHORS (LAST NAME, FIRST NAME): O' Dwyer, Ciara A.; Banville, Nessa; Egan, Sarah; McElvaney, Gerry; Reeves, Emer
INSTITUTIONS (ALL): Respiratory Research, Royal College of Surgeons in Ireland, Dublin, Ireland.
ABSTRACT BODY: Abstract Body: Cystic fibrosis (CF) is characterised by neutrophil-dominated lung inflammation attributable to release of the potent
neutrophil agonist, leukotriene B4 (LTB4). LTB4 is a pro-inflammatory agent that rapidly activates neutrophils through the G-protein coupled receptors BLT1 and
BLT2, causing release of proteolytic enzymes including neutrophil elastase which in turn can cause production of LTB4 by macrophages. Thus, there is a need for
therapies that target LTB4 thereby breaking this inflammatory cycle. The aim of this study was to investigate the ability of exogenous alpha-1 antitrypsin (AAT), an
anti-inflammatory circulating protein, to act as a LTB4 antagonist. The biological consequence of the AAT induced inhibition was investigated at the level of
calcium (Ca2+) flux and downstream activation events including neutrophil adhesion and release of proteolytic enzymes.
Circulating neutrophils were isolated from healthy control volunteers (n=15). Ca2+ and inositol triphosphate (IP3) levels were assessed fluorometrically by
stimulating cells with LTB4 (100nM) in the presence and absence of AAT (27.5µM). Neutrophils were loaded with calcein-AM dye and adherence to fibronectin
coated surfaces in response to LTB4 and LTB4 with AAT was examined. The effect of AAT on LTB4 induced neutrophil degranulation of primary, secondary and
tertiary granule components was analysed by stimulating neutrophils with LTB4 (25-200nM/ 2x107cells/mL) in the presence of increasing concentrations of AAT
(1.8-27.5μM) for increasing increments of time (0, 5, 10 or 20 min). The levels of released myeloperoxidase (MPO), hCAP-18, matrix metalloprotease 9 (MMP9), markers of 1o, 2o and 3o granule release respectively, to the surrounding supernatants were determined by Western blot analysis (n=6). The interaction
between LTB4 and AAT was analysed by UV absorption on a Jenway 6405.
Our in vitro data indicates that AAT prevents LTB4 signalling, as indicated by the lack of cytosolic Ca2+ flux and IP3 increase. Results demonstrate that the
adhesive effect of LTB4 is prevented in the presence of AAT (P<0.05) and that the levels of degranulated MPO, hCAP-18 and MMP-9 were significantly
decreased in the presence of AAT (P<0.05). The mechanism of inhibition involved direct binding of AAT to LTB4 as reduced vibrational fine structure of the
LTB4/AAT UV absorbance spectrum indicated complexation of the two molecules, with a binding affinity of 9.8 ±0.22 x 104 M-1. LTB4 appears to bind to the
hydrophobic pocket on the AAT molecule, as determined by competitive displacement assays with gemfibrozil (0.25μM), a hydrophobic lipid-lowering drug. The
interaction with LTB4 does not affect AAT from binding to and inhibiting its primary target neutrophil elastase.
The results of this study indicate that AAT can inhibit LTB4 signaling thereby reducing adhesion and the degranulation of proteolytic enzymes by neutrophils. This
study provides a novel therapy for targeting the elevated LTB4 levels present in the lungs of individuals with CF and thus proposes AAT aerosolized augmentation
therapy as an effective treatment for LTB4 associated pulmonary diseases.
Funding body: US Alpha-1 Foundation..