Idiopathic anaphylaxis (IA) was first described in 1978 in a series of patients with recurrent anaphylactic episodes where no specific trigger could be identified. IA usually manifests as urticaria, angioedema, wheezing, stridor, and, most importantly, may include hypotension, tachycardia, and sudden cardiac death. Although no offending allergens can be determined in patients with IA, mast cell degranulation and subsequent release of inflammatory mediators is thought to cause the disease. This hypothesis is corroborated by the elevated levels of mast cell-derived tryptase found in the serum of IA patients after an episode. ? The evidence for enhanced mast cell reactivity in IA is conflicting. An early study at NIH (Keffer et al. J Allergy Clin Immunol 1989) found no difference in the cutaneous response to either morphine or histamine in patients with IA compared to normal controls or patients with systemic mastocytosis. In contrast, a more recent study (2004) found that IA patients demonstrated higher skin responses to codeine than atopic controls. In mast cells and basophils, IgE receptor crosslinking leads to receptor phosphorylation by lyn, a src family kinase, which phosphorylates a second tyrosine kinase, syk. Phosphorylation and activation of syk leads to recruitment of numerous downstream signaling molecules such as phosphoinositide-3-kinase (PI3K), which catalyzes the formation of phosphatidylinositol-3, 4-5 phosphate (PIP3). PIP3 generation is critical to the release of calcium from intracellular stores leading to degranulation. So-called non releasing basophils and mast cells are deficient in syk. Conversely, some patients with hyper-releasable basophils were found to be deficient in src homology 2-containing inositol phosphatase (SHIP1), which dephosphorylates PIP3 and is thought to be a negative regulator of IgE-mediated signaling.? Agents acting on G-protein coupled receptors (GPCRs), such as histamine and morphine, induce mast cell and basophil degranulation independently of IgE. GPCRs activate heterotrimeric G proteins, which bind guanosine triphosphate (GTP) in exchange for guanosine diphosphate (GDP). The GTP-bound form of the G protein induces downstream signaling cascades, including intracellular calcium flux responsible for mast cell degranulation. In recent years, several compounds acting on GPCRs, such as chemokines or the serum factors sphingosine 1-phosphate and adenosine, have been shown to either activate mast cells themselves or to be required for optimal IgE-mediated degranulation. We have identified a regulator of G protein signaling (RGS13) expressed in mast cells, which appears to regulate both GPCR and IgE-mediated mast cell degranulation by distinct mechanisms. Mice deficient in RGS13 had markedly increased anaphlyactic responses due to more IgE-mediated mast cell degranulation. ? Although evidence from rodent models and the aforementioned patient data suggest that some signaling molecules profoundly influence mast cell reactivity, a systematic analysis of signaling components from patients with IA has not been performed. The goal of this project is to examine the IgE- and GPCR induced degranulation of mast cells grown in vitro from IA patients in comparison to allergic patients and normal controls and to evaluate the possibility of specific polymorphisms in RGS13 that might affect expression or function of the protein in mast cells. Such cells will be cultured from peripheral blood, and degranulation to IgE crosslinking and specific GPCR agonists will be examined. In addition, levels of natural GPCR ligands in patient sera will be assayed. Finally, the RGS13 gene will be sequenced. Preliminary analysis of mast cell RNA from a patient with severe allergies and increased IgE-mediated mast cell degranulation showed an alternately spliced RGS13 transcript containing a frameshift mutation. These mast cells also appear to express lower amounts of RGS13 protein than mast cells from healthy controls. If corroborated by analysis of more patients, these data may provide insight into how the signaling pathways leading to degranulation may be abnormal in allergy and/or IA.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Intramural Research (Z01)
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