Asthma and allergic diseases are multifactorial, complex diseases involving the innate and adaptive immune systems as well as environmental factors. Allergic asthma is characterized by airway hyperreactivity (AHR), inflammatory infiltrates containing eosinophilic leukocytes, lymphocytes, and mast cells, increased mucus secretion, and elevated serum IgE levels. Type 2 helper T lymphocytes (Th2) play a critical role in orchestrating allergic airway inflammation. Upon recognition of their cognate antigen, Th2 cells produce cytokines that regulate IgE synthesis (IL-4 and IL-13), and growth and activation of eosinophils (IL-5) and mast cells (IL-3 and IL-4). IL-13 and IL-9 control mucus production and AHR. Allergen-induced airway inflammation can be triggered by NKT cells and is antagonized by regulatory T (Treg) cells. Despite these progresses in our understanding of allergy pathogenesis and improved therapeutics, there remains a great deal to be learned to better control asthma and other allergic diseases. To attack these complex diseases, our approaches in investigation need to be multi-faceted. In this group project, we will investigate how allergic inflammation orchestrated by Th2 cells is regulated at cellular and molecular levels. To fulfill this common goal, we will study innate immune (dendritic cell and NKT cell) responses to an environmental factor, house dust extracts (HDEs), in Project 1. We will study how Th2 cells are regulated by protein kinase C? (PKC?) in Project 2, how different IgEs differentially affect mast cell activation in Project 3, and how Treg cells are regulated by Itch, an E3 ubiquitin ligase, in Project 4. Therefore, we will study the pathogenetic roles of the cells that belong to the innate and adaptive immune systems. The molecules we characterize with regard to allergy pathogenesis are widely varied, encompassing an environmental factor (HDEs), a secreted molecule (IgE), and intracellular signaling molecules (PKC9 and Itch). As described above, these cellular and molecular elements all likely contribute to various aspects of the pathogenesis of allergic asthma and other allergic diseases. Each component in this group can stand alone as an individual research project deals with an important, unique aspect of allergic diseases. However, cooperation among individual components will be multi-layered, e.g., exchanges of ideas, sharing reagents, protocols, and data, and formal and informal collaborations, to achieve our common goal of better understanding the diseases, just as allergic diseases involve various layers of extracellular, cellular, and intracellular elements. Upon completion of the program project, we will have gained a novel, unique, integrated set of insights into allergic diseases at all these layers. ? ? PROJECT 1: Desensitization of human mast cells: mechanisms and potential utility of attenuating asthma and allergic disease (Schwartz, L) ? ? PROJECT 1 DESCRIPTION (provided by applicant): Asthma and allergic diseases are important health concerns, and mast cells and possibly basophils are the major effector cells of IgE-mediated immediate hypersensitivity in humans. Previous work by the investigators involved in Project 1 includes developing tryptase as a clinical marker for mast cell activation; finding activation of mast cells in the asthmatic airway and during systemic anaphylaxis; identifying two types of human mast cells (MCT and MCTC) based on the protease content of their secretory granules and the surface expression of CD88; discovering the activating form of FcyRII, CD32a, constitutively expressed on the surface of skin MCTC cells; and finding Syk-deficiency in basophils with the non-releaser phenotype and in mast cells after antigen desensitization in vitro. The three specific aims of the current project are to: 1. Test the hypothesis that human mast cells and basophils in vitro undergo cross-desensitization to different antigens and heterologous desensitization through FcyRI and FcyRlla. Indeed, preliminary data suggest that when mast cells of the MCTC type are IgE anti-DNP-sensitized and then desensitized with low doses of DNP-BSA, both cross-desensitization and heterologous desensitization occurs. 2. Explore in vitro the mechanism(s) for desensitization of human mast cells involving Src and/or Syk tyrosine kinases. Although Syk depletion seems likely, the involvement of Lyn and/or Fyn is uncertain. Further, the possibility that subcellular compartmentalization of signaling differs for desensitization and activation will be examined. Collaborations with Project 3 on studies of Lyn kinase involvement in desensitization, and with Project 4 on the involvement of sphingosine kinase-1 in desensitization of human mast cells and basophils facilitate these mechanistic studies. 3. Determine whether penicillin desensitization of human subjects in vivo produces antigen cross-desensitization of mast cells and basophils and depletes Syk from peripheral blood basophils. This clinical study will begin to translate our in vitro findings to the in vivo situation. Clinical tolerance due to desensitization can be distinguished from that due to immunotherapy by its rapid induction (hours) and short persistence (days) once allergen administration ceases. We hypothesize that desensitization targets primarily mast cells and basophils. Understanding more precisely the characteristics of and the mechanism(s) behind desensitization will enable physicians to better utilize this approach to reduce mast cell/basophil-mediated contributions to asthma and allergic diseases. ? ? ?
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