Eosinophils play a key role in the pathogenesis of respiratory syncytial virus (RSV)- induced airway inflammation and epithelial damage, which may predispose to allergic sensitization and development of asthma. We have shown that: 1) RSV infection of airway epithelial cells induces adhesion-dependent, degranulation of eosinophils; 2) The integral membrane glycoprotein, polymeric Ig receptor (pIgR), is up-regulated on RSV-infected airway epithelial cells and, in its secreted form (secretory component, SC), is a potent stimulus for eosinophil degranulation; 3) RSV is able to infect directly human eosinophils, which induces MAP kinase activity, activation of the potent transcription factor NF-kappaB, and production of chemokines. We hypothesize that pIgR expression on epithelial cells, activation of the Raf-1-MEK-MAP kinase pathway and nuclear translocation of NF-kappaB are critical events in the eosinophil activation during RSV infection. The following specific aims are proposed: 1) To characterize the role of pIgR in eosinophil degranulation induced by RSV-infected airway epithelial. The role pIgR in RSV- induced eosinophil degranulation will be studied by blocking the process using a panel of neutralizing monoclonal antibodies that recognized different epitopes of the pIgR, and by co-culture assays of eosinophils with epithelial cells stably transfected with the human pIgR. 2) To investigate the activation of NF-kappaB in RSV- infected human eosinophils and to determine its role in chemokine gene expression. The NF-kappaB family members that are activated by eosinophils by RSV and the mechanisms of NF-kappaB antisense oligonucleotides or double- stranded competitor oligonucleotides will be employed to determine their effect on RANTES and IL-8 secretion. 3) To study the Raf-1-MEK-MAP kinase pathway in RSV-infected eosinophils and to assess its importance in NF-kappaB activation and chemokine production. The mechanisms of Raf-1 activation in RSV-infected eosinophils, will be studied by examining the involvement of tyrosine kinases, protein kinase C, and p21 ras molecules in the activation of Raf-1 kinase. We will test the hypothesis that NF-kappaB activation and chemokine production (RANTES and IL-8) in RSV-stimulated eosinophils are linked to the activation of the Raf-1-MEK-MAP kinase pathway by the use of specific kinase inhibitors, antisense oligonucleotides, and dominant-negative mutants. Identification of critical molecules involved in eosinophil functions will help in identifying specific inhibitors for treatment of acute RSV infection and for prevention of its sequelae, including asthma.
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