Modulation of signaling proteins in cultured mast cells from chronic idiopathic urticaria patients

Abstract

Rationale Although mast cells and basophils have been implicated in the generation of urticarial lesions, signaling changes in the FceRI pathway are unexplored. In the present study, we compared SHIP-1, SHIP-2 and Syk levels from basophils and mast cells (MC) cultured from the peripheral blood of Chronic Idiopathic Urticaria (CIU) subjects. Methods Peripheral blood obtained from CIU and normal donors was fractionated by density gradient centrifugation. Basophils were purified by negative selection. MC were cultured from positively selected CD34+ progenitors for 6-8 weeks using reagents provided by UCB-Pharma. Histamine release was determined by automated fluorimetry. Signaling proteins in whole cell SDS lysates were measured by quantitative Western blotting. Results CIU donors were characterized as anti-IgE responders (CIU-R) if their basophils released >10% total histamine content to 0.1 mg/ml anti-IgE whereas non-responder (CIU-NR) basophils released ≤10% histamine content. As compared to their basophils, MC of CIU-R donors had 3.2, 4.0 and 15.9 fold increases in SHIP-1, SHIP-2 and Syk protein, respectively (n=3). In contrast, CIU-NR donor MC demonstrated 2.0 and 8.9 fold increases in SHIP-2 and Syk, with a 0.3 fold decrease in SHIP-1 protein, relative to their basophils (n=4). The MC of CIU-R donors contained increased SHIP-1 and Syk but decreased SHIP-2 protein compared to either CIU-NR or normals. Conclusions The expression of signaling molecules implicated in the regulation of FceRI-induced degranulation are differentially modulated in CIU-R versus CIU-NR and normal MC, implying a genetic disposition in CD34+ progenitor cells to respond to a cytokine milieu.