In approximately 50 percent of allergic patients, there is a recrudescence of symptoms 6-12 hours after initial exposure to antigen termed the late phase reaction (LPR). The cells (basophils, eosinophils, monocytes), mediators (histamine and leukotrienes), and cytokines (IL-1beta, IL-5 and GM-CSF) found in the LPR mimic those found in the inflammation that perpetuates chronic allergic asthma. Previously, the investigators identified a histamine releasing factor (HRF) from in vivo LPR blister fluids that caused histamine release (HR) from human basophils. This release was dependent on the IgE molecules (termed IgE+) from a subpopulation of allergic patients. The investigators have subcloned this HRF. The cDNA codes for a 23 kD protein that has no homology with any known antigen, chemokine or interleukin. HRF did not show direct binding to IgE+ as evidenced by ELISA technology and affinity chromatography. The investigator's recent data demonstrate that HRF primes all basophils for HR, IL-4 and IL-13 protein secretion. Additionally, HRF causes a calcium flux, chemotaxis and enhances IL-8 production from human eosinophils. It also binds to a subset of monocytes. Pertussis toxin inhibits the initial phase of the HRF-induced calcium signal in eosinophils, which suggests involvement of a G protein. Additionally, rottlerin, a non-staurosporine-derived kinase inhibitor, distinguishes HRF-induced from anti-IgE-induced HR. Very recently, the investigators have shown specific binding of iodinated HRF to human eosinophils. They hypothesize that HRF is a major stimulus for cellular activation and perpetuation of the LPR, and, therefore, may play a role in the pathogenesis of asthma. This hypothesis will be tested by examining HRF-induced cytokine mRNA and protein production from cells. This will help to enumerate the behavioral characteristics of this novel cytokine. The investigators will also examine the mechanism of priming induced by HRF and compare this to IL-3, known enhancer of HR. They further hypothesize that HRF has a unique receptor on the cell surface. This receptor/ligand interaction will be characterized and the signal transduction events examined. Their radiolabeled binding studies will be optimized and Scatchard analysis on eosinophils, as well as other cell types will be performed. The investigators will compare the G alpha protein profile in eosinophls and basophils, and determine which G protein alpha subunit is activated upon HRF stimulation. The investigators will attempt to immunoprecipitate this receptor using their recombinant fusion protein.
