Chronic urticaria is a skin disorder characterized by whealflare lesions lasting less than 24-36 hours at a time, at least 4 timesweek, for more than 6 months. In some cases, defined physical stimuli such as pressure, vibration, heat, or cold induce urticarial episodes. The disease is termed chronic idiopathic urticaria (CIU) when no underlying cause (e.g. physical stimuli, atopy) can be identified. Skin lesions are characterized by an inflammatory cell infiltrate composed of CD4 T lymphocytes, eosinophils, neutrophils, and basophils. Because the urticarial lesions resemble the immediate skin lesions caused by mast cell degranulation after allergen injection as well as the basophilic late-phase skin response to allergen, a central function for mast cells and basophils in the pathogenesis of urticaria has been postulated. In physical urticarias, in vivo mast cell degranulation has been documented after exposure to the causative stimulus.? In recent years, an autoimmune etiology for CIU has been proposed. The basis for this hypothesis is the finding that nearly 40% of CIU patients have circulating autoantibodies, mostly toward the alpha subunit of the high affinity IgE receptor on mast cells and basophils or against IgE itself. In addition, another 40% of CIU patients have serum histamine releasing activity (HRA), which means that their sera can induce spontaneous histamine release from normal basophils. This serum factor has not been characterized, and may or may be attributable to the serum autoantibodies referred to above.? Although increased basophil (and presumably mast cell) activation by anti-IgE or anti-IgE receptor autoantibodies presents a simple and attractive etiology of CIU and possibly other urticarial disorders, there are several problems with this hypothesis. First, these serum autoantibodies are not specific to CIU. Anti-IgE and anti IgE receptor autoantibodies are found in a percentage of healthy subjects as well as in patients with other autoimmune diseases such as pemphigus vulgaris and dermatomyositis. Second,a majority of CIU patients lack either autoantibodies or HRA. Third, there is a poor correlation between basophil activation surface marker expression and either HRA or anti IgE receptor antibodies.? 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. In contrast to CIU, a study of IA patients found no evidence of IgE receptor autoantibodies. ? An alternative explanation to the autoantibody hypothesis involves abnormalities in the reactivity of basophils and mast cells in CIU and IA. Basophils isolated from some CIU patients exhibit reduced histamine release to IgE receptor crosslinking, which correlates with disease severity and increased expression of surface activation markers, such as CD63 and CD69. In contrast, CIU basophils release histamine normally to other factors such as ionomycin and N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Active CIU is accompanied by basopenia, presumably due to increased basophil migration into the skin. These results suggest that basophils (and possibly mast cells) may contain intrinsic defects in signal transduction pathways.? 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. Similarly, a study of cutaneous reactivity to compound 4880, which is thought to activate heterotrimeric G proteins, found an increased response in CIU patients with active disease compared to CIU patients in remission or normal 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 cross regulates both GPCR and IgE-mediated mast cell degranulation. Mice deficient in Rgs13 exhibit markedly increase anaphlyactic responses. ? Although evidence from rodent models and the aforementioned patient data suggest that these signaling molecules profoundly influence mast cell reactivity, a systematic analysis of signaling components from patients with either CIU or 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 CIU and IA patients in comparison to allergic patients and normal controls and to evaluate the possibility of specific mutations in RGS13 that might affect expression or function of the protein. Mast cells will be cultured from peripheral blood, and degranulation to IgE crosslinking and specific GPCR agonists will be examined. In addition, levels of GPCR ligands in patient sera will be assayed, and the RGS13 gene will be sequenced. These preliminary experiments data should provide insight into which specific signaling components may be abnormal in CIU andor IA. Eventually, specific in situ studies shall be performed to evaluate mast cell reactivity in these diseases based on in vitro data.
