MOLECULAR MECHANISMS OF GM-CSF PRODUCTION BY HUMAN EOSINOPHILS

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The mechanisms by which peripheral blood eosinophils (PBE) differentiate into airway based, effector cells responsible for the pathophysiology of asthma are poorly understood. A candidate mediator for this process is GM-CSF, a potent cytokine produced by activated eosinophils and other inflammatory cells. GM-CSF levels are elevated in the BAL fluid of active asthmatics while in vitro this cytokine has profound effects on eosinophil function and survival. Eosinophils activated in vitro with ionophore, fibronectin or VCAM-1 secrete GM-CSF and accumulate GM-CSF and accumulate GM-CSF mRNA. Thus, available data strongly suggest that GM-CSF is an important autocrine growth and survival factor for PBE function both in vitro and in vivo. Despite the likely functional significance of GM-CSF, very little is known about the molecular mechanism)s) which control its production and release by eosinophils. In T lymphocytes, fibroblasts and mast cells, mitogen induced GM-CSF production is dependent on both increased transcription and decreased mRNA degradation. Therefore, we hypothesize that similar, dual control mechanisms also modulate GM-CSF production from activated eosinophils. Such gene regulation studies have not been performed in eosinophils as they are difficult to transfect or isolate intact mRNA from. Utilizing particle mediated gene transfer (PMGT), we have successfully transfected resting peripheral blood eosinophils with luciferase reporter constructs. Secondly, we demonstrate that intact mRNA can be isolated from eosinophils and successfully hybridized with GM-CSF specific cDNA probes. Therefore, we are in a unique position to investigate the molecular mechanisms responsible for the elaboration of GM-CSF by activated eosinophils. This, our specific aims are: 1). Optimize the transfection of eosinophils by particle mediated gene transfer. 2). Determine the relative contributions of transcription stability to accumulation of GM-CSF mRNA in eosinophils after treatment with ionophore, fibronectin and VCAM-1. 3). Determine the mechanism of GM-CSF mRNA stabilization in activated eosinophils. 4). Identify the cis-trans interactions which control GM-CSF promoter upregulation in activated eosinophils. Cumulatively, these studies will closely interact with other components of the SCOR and provide novel information concerning the molecular control of GM-CSF production by activated eosinophils.