CYTOKINE REGULATION OF HUMAN EOSINOPHIL GENES

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Eosinophils are myeloid cells recognized to play a pivotal role as pro-inflammatory or cytotoxic effector cells in a wide variety of diseases of allergic, parasitic, neoplastic, and idiopathic origins. The proliferation and differentiation of these granulocytes in the bone marrow, and their post-mitotic functional maturation and activation in tissues occurs in response to a variety of growth factors and cytokines including GM-CSF, IL-3 and EL-5. While GM-CSF and IL-3 are responsible in part for the lineage commitment of granulocyte progenitors, including priming for certain eosinophil responses such as oxidative burst, lipid and protein mediator release, IL-5 is specifically involved in the differentiation and functional activation of eosinophils; all three cytokines support prolonged eosinophil survival and the development of an activated phenotype in vitro. Our specific aims are to investigate the mechanisms for molecular regulation of eosinophil genes in response to cytokine (IL3, IL-5) and growth factor (GM-CSF) mediated stimulation of normal eosinophil differentiation and post-mitotic functional activation. Using cDNA and genomic clones we have available for the eosinophil granule proteins including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), major basic protein (MBP), eosinophil peroxidase (EPO) and Charcot-Leyden crystal (CLC) protein, we will initially define the most appropriate eosinophil gene for study (i.e. the most eosinophil-specific and/or transcriptionally upregulated). We will focus on the regulation of only one or two genes in inducible eosinophil-committed leukemic cell lines, cytokine-driven differentiation of normal bone marrow or umbilical cord blood-derived progenitors, and cytokine priming/activation of eosinophils from the peripheral blood of normal donors or patients with eosinophilia. The objectives of this proposal are to use studies of eosinophil gene regulation to understand the processes involved in eosinophil lineage commitment, differentiation and priming/activation in response to specific cytokines and growth factors, and ultimately to identify eosinophil-specific transcription factors. The proposed research will address the following questions: 1) What are the mechanisms for the upregulation of the eosinophil granule protein genes? 2) What are the promoters and regulatory elements of eosinophil granule protein genes? 3) What changes in DNAse hypersensitivity are observed in these genes in myeloid (eosinophil) and non-myeloid cells and how are these changes cor-related with alterations in the expression of mRNA during the course of eosinophil differentiation or post-mitotic activation in response to specific cytokines? 4) What are the cis-acting DNA transcriptional control elements responsible for high level expression of eosinophil-specific genes during cytokine-induced differentiation or cell activation? 5) What are the constitutive or cytokine-induced transcription factors which may react with these control elements to regulate eosinophil gene expression? The proposed studies aim to provide a more integrated understanding of the molecular basis for the complex cytokine and growth factor-mediated processes that occur both during normal eosinophil differentiation and the functional activation of eosinophils in tissues in human disease.