Cell Movement Through a TH2-Conditioned Extracellular Matrix

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Our long-term goal is to understand how eosinophils (EOS) traffic to and interact in the ainway and contribute to the progression of asthma. aM32 integrin (CD11b/CD18) is highly activated on EOS obtained by bronchoalveolar lavage after segmental antigen challenge, suggesting that aM(32 functions becomes important as EOS extravasate to and migrate in the lung. However, little is known about roles of aMp2 and relevant ligand(s) in adhesion, migration, and activities of EOS in the airway. The overall objective of this proposal is to determine the role of aMp2 in modulating behavior of EOS in the ainway. Based on our preliminary data and current understanding of EOS biology and the extracellular matrix (ECM) in asthma, the current hypothesis is that periostin, an ECM protein characteristic of inflammation driven by T helper type 2 cells and found in the asthmatic ain/vay, is a dominant adhesive ligand for EOS aMp2 integrin and that the aMp2-periostin interaction is an important determinant of EOS function. Aim 1 is to define roles of aMp2, periostin and TGF-P-induced protein (TGFBI), a periostin homolog also found in lung, in adhesion, migration, sun/ival, and other functions of EOS activated by IL-3, IL-5, GM-CSF, or other activators. Aim 2 is to determine the structure-function relationship ofthe recognition of periostin by aMP2, map the aMp2-binding site(s), and define minimal constructs that when immobilized support, and when soluble block, EOS adhesion and migration. How the vitamin K-dependent y-carboxylation and alternative splicing affect periostin's biological activities will also be determined. Aim 3 is to understand the mechanism and significance ofthe striking increase of periostin that is found in the asthmatic airway. Periostin secretion from fibroblasts and epithelial cells stimulated by TGF-p or other factors, as well as its deposition into ECM and turnover will be analyzed. Antibodies to the various forms of periostin and TGFBI will be developed for localization in bronchial biopsies after segmental antigen challenge. Achieving the goals of this proposal will provide novel knowledge and a better understanding of EOS trafficking and functions and the interplay between EOS and the ECM in Th2-driven inflammation, and will generate agents and reagents that will allow this interplay to be studied and modulated. RELE^VANCE (See instructions): The proposed research will determine how the connective tissue protein periostin, which is strongly up- regulated in asthma, interacts with its cell-surface receptor protein aMP2 integrin (CD11b/CD18) on activated EOS, and supports attachment and migration of EOS. The project will provide new insights into the movement of EOS in the asthmatic ainway and the biology of connective tissue in the diseased lung.