Background: A common feature of inflammatory disease is the central role of Fc receptors and immune complexes and the high levels of cytokines produced, which serve to recruit other immune cells thus amplifying the response. Understanding how Fc receptors communicate intracellular signals to initiate cytokine responses and how the released cytokines contribute to inflammation is the major focus of our efforts. Our previous efforts have identified several proteins that are important in the regulation of gene expression initiated by IgE Fc receptor stimulation. We have focused on signaling proteins that may serve as possible links from Fc receptor to gene expression and mast cell degranulation. Our studies demonstrated the functional role of adaptor molecules like the linker for activation of T cells (LAT) and the Grb2-associated binder (Gab2). In the course of these studies we discovered the the IgE Fc receptor utilizes a second receptor-proximal Src family kinase for signal initiation. Fyn kinase was found to be essential for mast cell effector responses and some of its function is mediated through its role in phosphorylating Gab2 leading to activation of phosphatidylinositol 3-OH kinase (PI3K). These findings have provided a new paradigm in how activating Fc receptors couple to signal generation, which is essential for a cells response. Objectives: In the past year our objectives were as follows: 1. To investigate whether the coupling of the IgE Fc receptor to the generation of intracellular signals is influenced by the strength of the initial stimulus that engages the receptor. We engaged in this topic of investigation based on the observation that low affinity antigens were ineffective in producing a cellular response although they elicited receptor phosphorylation. 2. To investigate and dissect the relative importance of the adaptor molecules LAT and Gab2 in IgE Fc receptor responses. This line of investigation was based on our prior finding of two distinct molecular signaling complexes anchored by these adaptor proteins. 3. To investigate the importance of Lyn kinase in IgE Fc receptor-induced responses. This objective was based on our prior finding that Lyn is dispensable for IgE Fc-receptor activation of mast cells. Results: The objectives of the past year were met in the following manner. First, our studies on the influence of stimulus strength on functional coupling of Fc receptors revealed that engagement of low numbers of IgE Fc receptors is sufficient to elicit a potent cellular response. Thus, when only 10% of IgE Fc receptors on mast cells were engaged by IgE-antigen complexes we observed the production and secretion of chemokines and certain cytokines at levels that were very similar to that seen when 100% of the IgE Fc receptors are engaged. Moreover, in an effort to understand the underlying mechanism, we found that the adaptor Gab2 was more important for the observed chemokine response than the adaptor LAT. We also found that Fyn kinase is preferentially activated under these conditions. To understand the importance of the adaptor LAT, we studied the IgE Fc receptor-induced mast cell responses of cells expressing mutant forms of LAT in which the four carboxyl-terminal tyrosines were systematically mutated. These studies revealed that all four tyrosines were functionally important. However, mutation of the PLCgamma binding site showed a stong inhibitory effect on mast cell cytokine production and degranulation. In studying the relative importance of Fyn and Lyn kinases in the functional coupling of the IgE Fc receptor we found that Lyn kinase functions primarily as a negative regulator of allergic responses. Our findings showed that in the absence of Lyn kinase there is increased IgE production in vivo and mast cells become hyperresponsive. Our investigation also showed that mast cell hyperresponsiveness is partly due to enhanced activation of Fyn kinase. This is due to the loss of negative regulation of Src family kinases, in the absence of Lyn kinase, because of a failure to membrane target the C-terminal Src kinase, which is a critical for the inactivation of Src family kinases. Conclusions and Significance: In summary, we found that LAT is a key component of IgE Fc receptor stimulated responses through, in large part, its ability to bind and activate PLCgamma. However, LAT activation requires a stronger stimulus than the activation of Gab2 and thus IgE Fc receptor-mediated responses to a weak stimulus can be elicited through Gab2. The profile of lymphokines released suggests the possible initiation of allergic inflammation under these conditions. Finally, this work also demonstrate that the role of Lyn kinase in the allergic response is one of a negative regulator. Our studies point to a requirement of Lyn in controlling B cell mediated IgE production as well as mast cell responsiveness. In the coming year, we will focus on the relationship between Src family kinases and adaptor proteins in driving mast cell responses by identifying intracellular targets of their activity and their binding partners. We expect these new efforts will continue to provide important information for evaluation of the potential of these proteins as therapeutic targets for intervention in inflammatory disease.
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