This project focuses on the mechanism of activation of pi integrins on eosinophils (EOS) in the blood ofpatients with asthma; the role of such activation in selective movement of EOS into the airway; and'twophenomena that we hypothesize are important for movement of EOS within the airway, formation of podosomesand activation of aMp2 integrin.
Aim 1 addresses the hypothesis'that interaction of blood EOS Pselectinglycoprotein ligand (PSGL) with P-selectin, which is normally sequestered in a-granules of plateletsand Weibel-Palade bodies of endothelial cells, is responsible for enhanced p1 activation. This hypothesis willbe tested by correlative studies of blood samples obtained from patients with asthma of varying severity andasthmatic volunteers studied after whole lung or segmental lung antigen challenge. Measurements will bemade of the amounts of the N29 activation-sensitive epitope of pi on circulating EOS, P-selectin associatedwith circulating EOS, P-selectin on the surface of circulating platelets, and soluble P-selectin in plasma. Invitro studies will compare how the various forms of P-selectin enhance expression of the N29 epitope onEOS when added to blood and identify signaling pathways important for the increased expression.
Aim 2 addresses the hypothesis that activation of a4p1, the major pi integrin on EOS, primes the EOS to arrest invascular cell adhesion molecule (VCAM)-expressing vessels of asthmatic lung and thus enter the airway. Wewill correlate N29 epitope expression with various measures of eosinophilic inflammation in lung and relatethe time course of changes of N29 expression after lung Ag challenge vis-a-vis changes in P-selectin.Parallel in vitro studies will test if P-selectin enhances EOS adhesion from whole blood under static and flowconditions and identify signaling pathways important for the increased adhesion. The podosome is atransient structure that mediates interaction with and proteolysis of adhesive ligands in extracellular matrix.The podosome of EOS, which forms when cells stimulated with IL-5 and/or TNF-a adhere via a4p1 toVCAM, has a distinctive set of associated cytoskeletal and cell membrane proteins when compared topodosomes of other cell types. Included within this set is the ADAMS membrane metalloproteinase.
Aim 3 tests the hypotheses that podosomes and ADAMS contribute to the robust proteolytic capacity of adherentEOS; and that there is a 'hand-off1 when the EOS enter the lung such that aMp2, which is activated uponexposure to IL-5, replaces a4p1, which is degraded in podosomes, as the principal adhesive integrin. Theresearch will lead to a better understanding of trafficking of EOS in asthma and how trafficking may bemodulated pharmacologically.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
1P01HL088594-01A1
Application #
7391414
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
2007-12-01
Project End
2012-11-20
Budget Start
2008-02-01
Budget End
2009-01-31
Support Year
1
Fiscal Year
2008
Total Cost
$431,934
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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