Basophils and mast cells are the major carriers of high affinity receptors for IgE, and therefore are likely to be the major effector cells in immediate type allergic reactions. Both cell types undergo IgE- mediated exocytosis to release histamine and to also generate LTC4. For mast cells, the neutral protease called tryptase, when measured in blood or tissue lavage fluids, serves as a precise marker of mast cell activation, and antibodies against tryptase are sensitive and precise immunohistochemical probes for mast cells in tissues. Unfortunately, no such marker for basophils has been identified. Consequently, the presence and involvement of basophils at sites of inflammation, such as in the lung of asthmatics or the skin of atopic dermatitis patients, and the involvement of basophil activation in various forms of systemic anaphylaxis remain unresolved. The research proposed in this application will seek to identify and utilize basophil-specific proteins for assessing the involvement of this cell in human disease.
In Aim one, murine monoclonal antibodies will be generated against antigens in highly purified human basophils. Each antibody will be purified and evaluated for its specificity against basophils by also comparing its binding to eosinophils, neutrophils, lymphocytes and monocytes from peripheral blood, and mast cells in tissues and in culture.
In Aim two an initial characterization of each antigen corresponding to the antibodies in Aim one will be performed. this will include an analysis by Western blotting, gel filtration, and isoelectric focusing, and subcellular localization by immunoelectron microscopy and analysis of activated basophils.
In Aim three an immunoassay against a releasable, granule constituent of basophils will be developed to assess basophil activation in human disease.
