Lung allergies, in particular Bronchial Asthma, rank among the most common chronic conditions in the United States, affecting approximately 15 million Americans and causing serious illness and several thousands of deaths. The direct and indirect financial burden created by this disease runs in the billions of dollars. The symptoms underlying this disease involve IgE antibodies, infiltration of lungs by lymphocytes and eosinophils, secretion of mucous, and hyperactivity of the bronchial smooth muscle. Cytokines secreted by Th2 lymphocytes regulate the inflammatory response seen in allergic asthma. In particular, cytokines such as IIL-4, IL-5, and IL-13 are critical for the production of IgE antibodies, recruitment of eosinophils, and the subsequent release of inflammatory vasoactive amines and lipid mediators. The activation of the Inducible T cell Kinase, ITK, has been shown to regulate the production of Th2 cytokines. Interestingly, mice with disrupted ITK genes (ITK-KO mice) display significantly milder symptoms of lung inflammation, as assessed in an experimental model of bronchial asthma. The activation of ITK requires the presence of the adaptor protein SLP 76 and transphosphorylation by the Src kinase LCK. In the absence of SLP 76 ITK does not become transphosphorylated and thus remains enzymatically inactive. In vitro studies have uncovered an interaction between ITK's SH3 domain and the polyproline region (PR) of SLP 76. However, the biological significance of this interaction on ITK activation and subsequent cytokine production in live cells has not been determined. In the present investigation we will use competitive peptide inhibitors in an attempt to disrupt the association between ITK and SLP 76 and inhibit the activation of ITK in live cells. This presumably should create a condition that would mimic the situation with ITK-KO mice. To accomplish this we will assess the feasibility of a novel area of investigation for designing and testing cell-permeable synthetic peptides that represent the specific site in the PR region of SLP 76 with which ITK's SH3 domain interacts. Synthetic peptides representing this site will be rendered cationic by the addition of Arginine residues and tested for their ability to translocate into the cytoplasmic milieu. The effects of such peptides on ITK-SLP 76 association, ITK phosphorylation and enzymatic activity, as well as cytokine production (both Th1 and Th2) will be tested using both the Jurkat cell system and primary murine splenic T cells. In addition, we will inject the polyArg-SLP 76 peptides into mice and test their effects, as above, on splenic T cells ex vivo. Finally, we will test the effects of these peptides on a murine model of Bronchial Asthma. This proposal is novel and in spite of some risk, it has the potential to enhance health-related research and open avenues for the rationale design of drugs for the treatment and management of lung allergies.
Statement Allergic asthma is an immunologically based disease that involves inflammation of the airway. Allergic asthma is considered a serious public health problem worldwide with a significant economic and social impact. Here, we propose studies that may uncover new ways of controlling the mechanisms underlying the pathogenesis of allergic asthma.
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| Grasis, Juris A; Guimond, David M; Cam, Nicholas R et al. (2010) In vivo significance of ITK-SLP-76 interaction in cytokine production. Mol Cell Biol 30:3596-609 |
