Asthma and other allergic diseases are widespread health problems for industrialized nations. In order to develop therapeutic agents that lessen the severity of these diseases, the biological processes that drive allergy need to be defined. Allergic reactions are triggered by excessive helper T cell responses to antigens; thus these cells are attractive targets for new therapeutics. The long-term objective of this proposal is to define specific intracellular signaling events within helper T cells that control the production of allergy- promoting cytokines such as IL-4. We have identified an important role for the cyclosporine receptor cyclophilin A (CypA) in regulating helper T cell responses. CypA alters the structure of peptide bonds adjacent to proline residues, and may thus regulate protein function by inducing conformational changes. Mice lacking CypA develop inflammation that contains eosinophils and mast cells, two cell types that have key roles in allergic responses. Helper T cells from these mice overproduce IL-4 and other allergy- associated cytokines and show increased activation of the key signaling molecule phospholipase C-gamma1 (PLCgl). A known regulator of PLCgl is Itk,a tyrosine kinase that controls IL-4 expression. CypA interacts with a proline residue in Itk;this interaction promotes Itk self-association, which is postulated to downregulate Itk activity, and also inhibits contacts between Itk and other factors that promote PLCgl activation. Based on these findings, our central hypothesis is that CypA functions as represser of Itk activity, thus limiting IL-4 expression by helper T cells. To explore this hypothesis, we will pursue the following specific aims: 1) Identify amino acids that are required for interaction between Itk and CypA and for Itk self- association;biochemical assays will be used to assess how amino acid changes in Itk and CypA affect protein-protein interactions;2) Determine the effects of mutations in CypA and Itk on function;CypA and Itk mutant proteins with altered function will be expressed in helper T cells to assess their impact onIL-4 expression and PLCgl activation;3) Determine the role of CypA and Itk in allergic disease;how changes in CypA or Itk activity modulate asthma development in mouse model for this disease will be analyzed;4) Determine whether CypA regulates the development of helper T cells that express allergy-promoting cytokines;a cell culture system will be used to define regulatory targets for CypA.
