The objective of this proposal is to purify and characterize a novel cytokine produced by human mononuclear cells that inhibits histamine release from basophils and mast cells. This histamine release inhibitory factor (HRIF) specifically antagonizes the activity of histamine releasing factors (HRF). The latter cytokines are produced by a variety of cells and evidence has been growing that they are involved in the pathogenesis of bronchial asthma and other allergic disorders. As an antagonist of HRF, HRIF will have immense biological importance in regulating allergic diseases. This cytokine will be generated in large quantities by culture of mononuclear cells purified from buffy coats obtained from Blood Bank donors, as well as U937 cell line. Preliminary experiments suggest that HRIF can be purified by ion exchange and size exclusion HPLC. The degree of purification will be judged by one and two dimensional polyacrylamide gel electrophoresis. The purified material will be analyzed for amino acid composition and sequencing. The long term objective is to construct a probe on basis of partial sequence data and clone the gene. In order to establish a quantitative assay of HRIF, monoclonal antibodies will be raised. This will be accomplished by immunizing mice with purified HRIF and by hybridizing the spleen cells with a myeloma cell line. Purified monoclonal antibodies will be used in the development of immunoaffinity columns, as well as ELISA or RIA. HRIF is produced by 8 cells, T cells and monocytes-macrophages. Biochemical and functional characterization of HRIF produced by different cell type will be performed by HPLC, gel electrophoresis and monoclonal antibodies. The activity of HRIF will be tested on human peripheral blood basophils, bronchoalveolar lavage mast cells, rodent peritoneal mast cells and HL 60 cell line. Additionally, the efficacy of purified HRIF to block allergic reactions in vivo will be assessed in rodents and guinea pigs. The clinical significance of HRIF will be evaluated by comparing the production of HRIF by mononuclear cells from normal subjects, allergic and asthmatic patients not receiving immunotherapy and allergic patients receiving immunotherapy. The effect of immunotherapy on the production of HRIF will be additionally determined in a prospective controlled study.
Showing the most recent 10 out of 18 publications
