The objective of this proposal is to characterize the soluble factors released by mononuclear cells that regulate the function of basophils and mast cells. These cells have a major role in immediate hypersensitivity reactions (asthma, rhinitis, anaphylaxis) caused by IgE antibodies. Basophils also accumulate locally in many other late onset reactions: contact dermatitis; rejection of tumors, microbes, transplants; and the chronic stage of IgE dependent reactions. It is postulated that mononuclear cells release cytokines that direct the migration of basophils and the secretion of basophils and mast cells. We have identified and characterized two human cytokines acting on basophils, a basophil chemotactic activity and a histamine-releasing activity. These factors will be prepared in large quantities by culture of leukocyte concentrates from normal blood donors and human T-cell lines. Factors will be purified by ion exchange, gel filtration, affinity and high performance liquid chromatographies. The cytokines will then be used for production of specific antibodies and for determining their effects on basophils and mast cells. Polyclonal antibodies will be prepared by immunizing rabbits with purified HRA in order to provide an assay for monoclonal antibodies to be prepared in mice. These antibodies will be used for affinity chromatography, characterization of cytokines, and measurements of HRA in biological fluids. The cytokines produced by unfractionated mononuclear cells will be compared with the products of purified T-cells, T-cell subsets and monocyte-macrophages. Comparisons to be made are physicochemical properties, antigenic cross-reactivity and functional response of basophils. One hypothesis to be tested is whether the same molecules can regulate both migration and secretion of basophils. We speculate that at low concentrations, cytokines induce cell movement, and at high concentrations, exocytosis. This will be tested by combining different concentrations of cytokines with solid or fluid phase antibodies, and then determining the function (chemotaxis and release of histamine) and morphologic (electron microscopy) responses of purified basophils. The biological significance of HRA will be studied by seeking this cytokine in blister fluid from patients with contact dermatitis, and in bronchoalveolar lavage fluid from asthmatics. The spontaneous and antigen-induced synthesis of HRA by cells from asthmatics cultured in vitro will be followed and levels correlated with the clinical course and response to immunotherapy.
Showing the most recent 10 out of 19 publications
