Investigator: Deborah J. Stearns-Kurosawa, Ph.D.
Location: Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma
Timeline: March 1, 2003 – February 29, 2004 (extended to June 2004)

Abstract
Vascular inflammation is a hallmark of Wegener’s Granulomatosis (WG) and other primary systemic vasculitides. Vascular inflammation is also characteristic of atherosclerosis, and WG patients have increased intimal thickening, more frequent cardiovascular disease and accelerated atherosclerosis. The concurrent endothelial dysfunction and altered flow hemodynamics will induce homing and transmigration of pro-inflammatory cells, including neutrophils.

Enzymes, cytokines and receptors expressed by emigrating neutrophils contribute to the ongoing inflammation. Neutrophils express proteinase-3, an enzyme located in neutrophil granules and recognized by the autoantibody in WG patients (PR3-ANCA). In vitro experiments have demonstrated that this PR3-autoantibody interaction induces a myriad of neutrophil inflammatory events. The in vivo importance of PR3-ANCA remains controversial in part because it is not clear how PR3 becomes accessible to the autoantibodies on largely unactivated neutrophils.

However, few investigators have considered potential contributions from flow-induced shear stress. The current data demonstrate that low level shear stress results in unique surface expression of PR3 on neutrophils; other neutrophil granule markers are largely unaffected.

Our hypothesis is that low shear stress levels are important for expression of neutrophil PR3, so that it becomes accessible to pro-inflammatory PR3-ANCA. We also hypothesize that this low level shear-induced neutrophil PR3 expression plays a major role in the localization of endothelial injury, since Wegener’s granulomatosis selectively affects small-to-medium sized vessels. Experiments also are proposed to identify shear-induced signaling mechanisms to provide mechanistic insight into PR3 expression under rheologic conditions.

Collectively, these events would bring neutrophils and their expressed PR3 and PR3/PR3-ANCA complexes in close proximity to endothelial cells, providing all the players to initiate or propagate inflammatory events.

Results from proposed studies will form the foundation of future studies identifying the molecular mechanisms underlying this unique influence of shear stress on neutrophil PR3 expression.

Specific aims
These aims are designed as a 1 year project to identify the rheologic conditions that result in surface PR3 expression on neutrophils exposed to laminar shear forces and on neutrophils rolling on endothelial cells under flow. Signaling mechanisms relevant to shear-induced neutrophil PR3 expression will be explored. Additionally, the ability of macromolecules known to bind to neutrophil PR3 under static conditions, including PR3-ANCA, will be determined under flow conditions.

1. Define the laminar flow conditions that result in neutrophil PR3 expression. Neutrophils will be exposed to varying levels of laminar shear stress in a parallel plate flow chamber. Surface PR3 expression will be evaluated by flow cytometry using anti-PR3 monoclonal antibodies and patient PR3-ANCA. These results will give insight into the influence of shear stress on PR3 expression and accessibility. Other granule markers will be evaluated concurrently. The binding of molecules known to interact with PR3 (e.g., alpha 1-antitrypsin, soluble endothelial protein C receptor) will be determined similarly under flow conditions for comparison with known static systems.
2. Evaluate the impact of shear stress and activated endothelium on PR3 expression on rolling neutrophils. Neutrophils will be rolled over TNF-α activated endothelial cells at varying physiological flow velocities and PR3 expression quantitated by flow cytometry.
3. The influence of modulators (patient PR3-ANCA, a1-antitrypsin, etc.) on adhesion, rolling velocities and transmigration will be determined. Neutrophils will be harvested for evaluation of signaling events using western blotting and ribonuclease protection assays. These studies provide a physiological context in which to evaluate the influence of laminar flow forces on PR3 expression, PR3 accessibility to PR3-ANCAs, and subsequent neutrophil adhesion events.

What it means for patients
No analysis of the type proposed has ever been attempted and a body of important new information will be developed as a result of this study. The approaches described in this proposal are new and unique ways to look at the fundamental mechanisms underlying the pathogenesis of WG and related vasculitides. The long-term consequences are impossible to predict, but knowledge of mechanisms regulating our response to inflammation may provide insight into novel therapies.

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