The synthesis of cysteinyl leukotrienes (CysLT)s by eosinophils plays an important role in the pathogenesis of asthma. We have discovered that secreted PLA2 group X (sPLA2-X) is involved in eosinophil CysLT synthesis, contradicting the long-held view that cytosolic PLA2? (cPLA2?) is the only major PLA2 involved in CysLT formation in eosinophils. Our overall hypothesis is that sPLA2-X is upregulated in eosinophils during inflammation of the airways and plays an important role in CysLT formation by eosinophils. We have recently demonstrated that exogenous sPLA2-X initiates CysLT synthesis by human eosinophils through a mechanism that is dependent upon the enzymatic function of sPLA2-X, but also involves the activation of cPLA2?, through p38 and c-Jun-terminal kinase (JNK). In preliminary data, we demonstrate that sPLA2-X is endogenously expressed in human eosinophils and that a highly selective inhibitor of sPLA2-X attenuates CysLT formation by fMLP-stimulated eosinophils. In the first specific aim, we will examine the intracellular location of sPLA2-X in human eosinophils using confocal microscopy. We will determine if the expression of sPLA2-X in eosinophils is increased by IL-3, IL-5, GMCSF and other cytokines that "prime" eosinophils for activation as they enter the airways. In a translational portion of this study, we will determine if sPLA2-X expression is increased in eosinophils from patients with asthma. In the second specific aim, we will examine the function of sPLA2-X in eosinophils first through selective inhibitors of sPLA2-X and cPLA2?, and then through lentiviral-mediated shRNA knockdown of sPLA2-X in the eosinophil-like cell line HL-60 clone-15. Since sPLA2-X can act within cells and after secretion, we will determine if sPLA2-X co-localizes with 5-lipoxygenase (5-LO) during activation, and if it is released during piecemeal degranulation, allowing the enzyme to act on the outer cell membrane. Finally, we will examine the potential function of sPLA2-X in cell-free eosinophil granules that act as secretion competent organelles infiltrating the airway tissues of patients with asthma. Establishing the important function of sPLA2-X in eosinophil CysLT formation will provide a strong rationale for this enzyme as a therapeutic target in asthma.

Public Health Relevance

Asthma is one of the most important public health problems in young adults in the United States. This study examines the function of a novel enzyme that may serve as a key regulator of inflammatory lipid mediator formation in asthma. The results will provide a strong rationale for this enzyme as a therapeutic target in asthma.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21HL111845-02
Application #
8522225
Study Section
Special Emphasis Panel (ZRG1-CVRS-J (03))
Program Officer
Noel, Patricia
Project Start
2012-08-15
Project End
2014-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
2
Fiscal Year
2013
Total Cost
$183,855
Indirect Cost
$64,855
Name
University of Washington
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195