Interleukin 4 is a pleiotropic cytokine that exerts its diverse effects on a wide variety of target cells of hematopoietic and non-hematopoietic lineage. IL-4 plays an important physiologic role in hematopoietic cell growth and differentiation both directly by influencing differentiation processes and indirectly by modulating the production of other hematopoietic growth factors such as IL-1 and TNF-alpha. It is a critical immunoregulatory molecule and modulates the inflammatory response, in part, due to its ability to affect adhesion molecule expression and cytokine production by endothelial cells and its ability to affect growth and/or activation state of neutrophils, mast cells, T cells and eosinophils. Dysregulated IL-4 production has been shown to result in pathological conditions such as atopic disease. The physiologic production of IL-4 is tightly regulated: it is expressed only by a subset of activated T cells and mast cells/basophils. Based on their different tissue distribution and access to distinct target cells, T and mast cell derived IL-4 may have quite different effects on physiologic/immunologic processes. Recently, information regarding regulatory elements that dictate IL-4 gene transcription in T cells has been reported. However, very little is known about regulation of IL-4 in mast cells. The long term goals of this proposal are to define the molecular basis of the cell specific-regulation of IL-4. With this information, strategies to selectively modulate its expression in a cell specific manner can ultimately be devised and used therapeutically. Dr. Brown has defined a key IL-4 regulatory element (ARE) that is a target for activation signals leading to IL-4 transcription in both T and mast cells. There are clear differences in the T and mast cell transcription factors that interact with this element. Clearly, the next step in the analysis of this region is identification and comparison of these component proteins. The experiments in this proposal are designed to assess the cell specific differences in IL-4 regulation mediated by the ARE and other IL-4 regulatory regions. Specifically, the following is proposed: (1) To compare the transcription factors that directly associate with an IL-4 activation responsive element in T and mast cells: T cell ARE binding proteins will be purified by affinity chromatography and microsequence information will be used to design oligonucleotides to screen an activated T cell cDNA library. The cDNA(s) identified will be characterized and used as probes to assess their expression in mast cells. (2) To compare T and mast cell ARE transcription factors that require cooperative binding or interact solely through protein-protein interactions for ARE association: A two hybrid cloning system that is specifically designed to detect interacting proteins will be used to clone those proteins that do not directly bind the ARE. The interacting factors in T and mast cells can be compared. (3) To examine the potential of other IL-4 regulatory elements, defined in T cells, to modulate mast cell IL-4 gene transcription: Mutations in defined T cell IL-4 regulatory elements will be introduced into 5' IL-4 sequences and their effect on the expression of linked CAT reporter gene will be assessed in mast cells.
NIH ExPorter
NIH ExPorter