Eosinophils are one of the key effector cells in the pathology of asthma. Activation of eosinophils by inflammatory mediators leads to the release of toxic granule proteins that cause serious damage to the airway epithelium. However, the post-receptor signaling pathways that control eosinophil degranulation and granule protein toxicity are poorly understood. The overall objective of this project constitutes the first specific goal of this AADRC, which is to understand the cellular and molecular mechanisms of eosinophil activation as they relate to the release of granule proteins. The long-term goal of Project 1 is to understand how calcium mobilization and PKC activation contribute to degranulation and to the toxic effects of granule proteins on the airway epithelium. The first specific aim of the project is to identify the PKC isoform involved in IL-5 stimulated degranulation and to investigate its role in regulation of cytoskeletal rearrangements necessary for degranulation. We will test the hypothesis that activation of PKC-delta, the major calcium-independent PKC isoform in human eosinophils, phosphorylates MARCKS, resulting in actin rearrangements that are critical for degranulation.
The second aim will address the effects of calcium mobilization on intra-granule acidification and examine the role of proton transport in granule protein solubilization. Our hypothesis is that increases in intracellular calcium stimulate granule membrane proton ATPases leading to granule acidification and solubilization of MBP crystals, a process needed for effective release of this protein. We further contend that MBP crystal solubilization strongly enhances toxicity resulting in a greater degree of epithelia damage compared to MBP release in the absence of increased intracellular calcium. To test this hypothesis, we will investigate the effects of calcium on granule membrane proton transport and measure the effects on MBP solubilization. Finally, we will examine the effects of eosinophil degranulation on human airway epithelial cell and determine the toxicity of released granule proteins on epithelia barrier function, electrolyte transport and mucosal shedding. We will use this system to evaluate effects of MBP solubilization on the airway epithelium and to determine whether compounds that inhibit granule acidification can reduce MBP toxicity. The results should provide new insights into eosinophil degranulation and elucidate the regulatory mechanisms of granule protein toxicity on airway epithelial cells. Furthermore, our findings may help identify new drug targets and therapeutic approaches for the treatment of human asthma.
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