The initiation and persistence of inflammation is tightly regulated by cell surface receptors on immune cells that function to both amplify the immune response and clear the offending agent. The persistence of inflammation may result in inflammatory disease inclusive of autoimmunity, chronic allergy and asthma. One common feature of these diseases is the central role of Fc receptors and immune complexes. A second common feature is the high levels of cytokines that serve to recruit other immune cells amplifying the response. Understanding how Fc receptors communicate with the nucleus to initiate cytokine responses and how the released cytokines contribute to inflammation is the major focus of our efforts. The immediate objective is to identify the molecular events that activate cytokine gene transcription and to evaluate whether these events might be suitable therapeutic targets for intervention of disease. We chose to work with mast cells because they are key participants in many inflammatory responses, they express the well-characterized high affinity IgE Fc receptor, and immune complex occupation of this receptor results in the production and secretion of a diverse array of cytokines. In the past year our objectives were: 1. To study the role of various signaling proteins in Fc receptor-induction of cell responses. 2. To determine in what signaling pathways the studied proteins participate. 3. To determine how critical these proteins are in induction of cytokine gene expression and cell function in response to stimulation of the IgE Fc receptor. We met these objectives in the following manner: We studied the function of the guanine nucleotide exchange factor Vav1 and its role in the regulation of IL-2 production. Our findings showed that Vav1 is translocated to the nucleus and that this translocation is regulated by an internal nuclear localization signal which is normally masked by the c-terminal SH3 domain of Vav1. When Vav1 translocates to the nucleus it facilitates the translocation of the transcription factor NFAT, whose activity is critical for induction of IL-2 gene expression as the IL-2 promoter contains an NFAT binding site. Moreover, the studies showed that Vav1 forms a complex with transcriptionally active NFAT and NFkB and that in the absence of Vav1 translocation or formation of a transcriptional complex with NFAT, IL-2 production is severely impaired. These studies demonstrate that Vav1 is a critical component of the transcriptional complex that drives IL-2 production and that its role in this complex is important as Vav1-deficient mast cells showed a severe defect in IL-2 production. We have also discovered that Fc receptors utilize Fyn kinase for their function. It was previously thought that all events downstream of IgE Fc receptor aggregation depended on the activity of Lyn and Syk kinases and the assembly of a LAT organized signaling complex. Our present findings demonstrate that signals can be generated that are independent of the well-characterized Lyn-Syk-LAT axis. Thus, we found that the IgE Fc receptor activates both Lyn and Fyn and that these two Src kinases initiate two independent pathways following IgE Fc receptor aggregation. We evaluated the importance of Fyn kinase in mast cell responses and found that gene-disrupted mice were resisitant to an anaphylaxis challenge and that mast cells from these mice showed severe inhibition of the degranulation response. Because the proteins under study are hematopoietic cell-specific and thus may be more suitable as possible therapeutic targets we will continue to explore their function by defining the regions of these proteins critical for the observed activity. Additionally, we will identify other proteins that interact with the studied proteins that may contribute to their function, thus providing further important information for evaluation of their potential as therapeutic targets for disease intervention.
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