Mast cells(MCs), basophils, eosinophils, and lymphocytes are integral to the development of an allergic response. Degranulation of MCs and granulocytes, and cytokine production by T cells is induced primarily by cross-linking of the receptor for antigen. However, allergic inflammation may also be generated through activation of receptors coupled to heterotrimeric G proteins (GPCRs). The purpose of this study is to understand mechanisms of intracellular G-protein-coupled signal transduction in immune cells and subsequent pathways to inflammation. GPCRs activate heterotrimeric G proteins, which bind guanosine triphosphate (GTP) in exchange for guanosine diphosphate (GDP). The GTP-bound form of the G protein alpha subunit induces downstream signaling cascades, including intracellular calcium flux responsible for MC/basophil degranulation. This project focuses on a family of regulators of G protein signaling (RGS proteins), which inhibit the function of G alpha-i and G alpha-q, but not G alpha-s, proteins by increasing their GTPase activity. G alpha subunits oscillate between GDP- (inactive) and GTP- (active) bound forms based on ligand occupancy of the associated receptor. The GTPase accelerating (GAP) activity of RGS proteins limits the time of interaction of active G-alpha and its effectors, resulting in desensitization of GCPR signaling. Despite a growing body of knowledge concerning the biochemical mechanisms of RGS action, relatively little is known about the physiological role of these proteins in allergic inflammation. Compounds acting on GPCRs, such as platelet-activating factor (PAF) and adenosine, induce granulocyte and MC degranulation independently of IgE. In previous years'work, we identified an RGS protein, RGS13, which inhibits IgE-mediated mast cell degranulation and anaphylaxis in mice by binding to and counteracting activation of the critical downstream enzyme phosphoinositide-3 kinase (PI3 kinase). We also found that RGS13 regulates GPCR-induced degranulation and cytokine production by human MCs through its GAP activity. These results uncovered a new physiological function of RGS proteins with broad implications for cell growth, metabolism, and immunity: the direct inhibition of PI3 kinase. We hypothesized that abnormalities in RGS13 activity may exist in patients with anaphylaxis or other disorders that may be associated with increased mast cell reactivity. Current studies of RGS13 have focused on the regulation of its expression in immune cells. We found that the tumor suppressor p53 binds to a functional site in the RGS13 promoter, leading to inhibition of RGS13 expression in MCs and B lymphocytes. During the course of these studies, we also discovered (using MCs from p53-deficient mice) that p53 exerts a negative regulatory effect on MC degranulation but is required for IgE-antigen-induced cytokine production. These results suggest that p53 expression and/or function should also be examined in clinical disorders of MC degranulation such as anaphylaxis. We also found that RGS13 was phosphorylated by protein kinase A (PKA). GPCR ligands such as beta-adrenergic agonists generate intracellular cyclic adenosine monophosphate (cAMP), which in turn activates PKA. We mapped the site of RGS13 phosphorylation by PKA and showed that phosphorylated RGS13 protein had a prolonged half-life inside cells. Collectively, these studies have provided insight into the regulation of RGS13 expression, and we have begun to search for polymorphisms affecting RGS13 expression and/or function in subjects with anaphylaxis. The other major area of investigation in this project is the recruitment of inflammatory cells to sites of allergic inflammation. A major class of compounds acting on GPCRs in leukocytes are chemokines. Chemokines and their receptors orchestrate cell trafficking during the immune response. We found that RGS16 was expressed in activated Th1, Th2, and Th17 CD4+ effector T cells. RGS16-deficient T cells migrated more to Th2-associated chemokines such as CCL17 in vitro. These results translated into abnormal T cell trafficking in Th2-associated pathologies. In collaboration with Dr. Thomas Wynn (Laboratory of Parasitic Diseases, NIAID), we showed that lungs from Rgs16-/- mice infected with the helminth Schistosoma mansoni had more inflammation, fibrosis, and T cell infiltration than wild type counterparts. We concluded that RGS16 attenuates Th2 responses to Schistosoma antigens. We have also embarked on identification of chemokine receptors, G protein, and RGS proteins involved in regulating the function of basophils in allergic inflammation in mice. Sensitization to protease allergens, such as papain, or helminth infection, is associated with basophil recruitment to draining lymph nodes. Basophils may promote Th2 differentiation directly through antigen presentation or indirectly through IL-4 production. How papain induces basophil migration to lymph nodes is unknown. We found that papain directly activates a subset of naive T cells that expresses protease-activated receptor 2 (PAR2). Papain induced PAR2+CD4+ T cell production of CCL17, CCL22, and IL-4 upstream of basophils by activating p38 mitogen activated protein (MAP) kinase and Jak3-STAT signaling pathways. Papain-triggered accumulation of CCL17, CCL22, and basophils in lymph nodes was abolished by the absence of CD4+ T cells or PAR2, and basophil trafficking was strongly diminished by CCR4 deficiency. These results defined a novel innate function of naive T cells in the direct recognition of a cysteine protease allergen through PAR2, which precedes TH2 effector cell development. As many environmental allergens have proteolytic activty, these findings have broad implications for the allergic response and suggest viability of therapeutic targeting of PAR2 in allergic diseases.
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