G-Protein-Coupled Signal Transduction in Allergy and Anaphylaxis

Druey, Kirk

Abstract

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. 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. This phenotype was due to inhibition of IgE-Ag induced PI3 kinase activation by RGS13. In 2009, we extended these findings by showing that RGS13 also regulates GPCR-induced degranulation and cytokine production by human mast cells. HMC-1 and LAD-2 mast cell lines depleted of RGS13 by shRNA showed increased calcium influx in response to several endogenous ligands such as C5a, sphingosine-1-phosphate, and the chemokine CXCL12. LAD-2 cells with reduced RGS13 expression degranulated more to sphingosine-1-phosphate than control cells. 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 goals of this project are 1) 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 (in collaboration with the Mast Cell Biology Section of LAD) and 2) to analyze the occurrence and functional significance of specific polymorphisms in the RGS13 gene (in germline DNA and RNA derived from cultured mast cells). These studies may provide insight into how signaling pathways leading to mast cell degranulation differ in IA from healthy controls.