Mechanisms and Consequences of Eosinophil Integrin Activation

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This project focuses on the mechanism of activation of pi integrins on eosinophils (EOS) in the blood of patients with asthma; the role of such activation in selective movement of EOS into the airway; and'two phenomena that we hypothesize are important for movement of EOS within the airway, formation of podosomes and activation of aMp2 integrin. Aim 1 addresses the hypothesis'that interaction of blood EOS Pselectin glycoprotein ligand (PSGL) with P-selectin, which is normally sequestered in a-granules of platelets and Weibel-Palade bodies of endothelial cells, is responsible for enhanced p1 activation. This hypothesis will be tested by correlative studies of blood samples obtained from patients with asthma of varying severity and asthmatic volunteers studied after whole lung or segmental lung antigen challenge. Measurements will be made of the amounts of the N29 activation-sensitive epitope of pi on circulating EOS, P-selectin associated with circulating EOS, P-selectin on the surface of circulating platelets, and soluble P-selectin in plasma. In vitro studies will compare how the various forms of P-selectin enhance expression of the N29 epitope on EOS 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 in vascular cell adhesion molecule (VCAM)-expressing vessels of asthmatic lung and thus enter the airway. We will correlate N29 epitope expression with various measures of eosinophilic inflammation in lung and relate the 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 flow conditions and identify signaling pathways important for the increased adhesion. The podosome is a transient 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 to VCAM, has a distinctive set of associated cytoskeletal and cell membrane proteins when compared to podosomes 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 adherent EOS; and that there is a "hand-off1 when the EOS enter the lung such that aMp2, which is activated upon exposure to IL-5, replaces a4p1, which is degraded in podosomes, as the principal adhesive integrin. The research will lead to a better understanding of trafficking of EOS in asthma and how trafficking may be modulated pharmacologically.