Eosinophils are known to be important effector cells in the pathogenesis of asthma. The cytokines interleukin-3 (IL-3), IL-5, and granulocyte- macrophage colony-stimulating factor (GM-CSF) are important in eosinophil development and activation. Without the influence of these cytokines, eosinophils rapidly undergo apoptosis. We have confirmed previous observations that eosinophils recruited to the airways of humans by segmental antigen challenge (SAC) show a prolonged survival ex vivo, in comparison with peripheral blood eosinophils. Moreover, we have recently determined that the prolonged survival appears to be primarily due to the autocoid secretion of GM-CSF by the airway eosinophils ex vivo. Investigations into the molecular mechanisms involved in apoptosis have exploded in the last several years. The Ced- 3/ICE cysteine protease gene family are important pro-apoptotic mediators, while Bcl-2 and related proteins provide important counter- regulatory effects and inhibit apoptosis. We have recently identified four novel isoforms of ICE (beta, gamma, delta epsilon) as well as the novel Ced-3/ICE homologs, CPP32, Mch2 and Mch3. Significantly, we have also identified several of these proteins in BAL eosinophils from asthmatics. In this investigation we propose to study the role of cysteine proteases in programmed cell death in eosinophils. We will first characterize the known cysteine protease genes and their products in BAL and peripheral blood eosinophils, and the eosinophil inducible cell line AML14. Second, we will determine the effects of glucocorticoids and the eosinophil-active cytokines on cysteine protease gene expression, and also systematically investigate mechanisms by which the eosinophil-active cytokines might antagonize glucocorticoid mediated apoptosis. Third, the effect of crosslinking eosinophil surface APO- 1/FAS-antigen on cysteine protease gene expression will be examined, as well as determining the role of apoptotic inhibitors BcL-2 and p35 on glucocorticoid- and FAS-mediated apoptosis. Finally, we propose a strategy for identifying and cloning novel apoptotic cysteine proteases from eosinophils and the AML cell line. The elucidation of mechanisms by which eosinophil survival is regulated in vivo at sites on inflammation is critical to our understanding of asthma pathogenesis. Furthermore, manipulation of these pathways will be a key area for therapeutic intervention in asthma.
