Research Article

Mast Cell Subsets and Their Functional Modulation by the Acanthocheilonema viteae Product ES-62

Figure 5

ES-62 modulates signalling in mast cell subsets. PDMC were sensitised with murine anti-DNP IgE (0.5 μg/mL) in the presence or absence of ES-62 (2 μg/mL) overnight. Following loading with Fura-2/AM such PDMC were stimulated at 50 s with DNP (0.5 μg/mL) to induce cross-linking (XL) of FcεR1 (a) or 0.5 μg/mL LPS (b) and intracellular calcium mobilisation and influx recorded in real time using excitation-emission ratios of 340/380 nm (a) & (b). Calcium levels were calculated from and values and data are presented as the mean calcium values of triplicate samples from a single experiment representative of at least 3 independent experiments. PDMC and mucosal-type BMMC (c) & (e) were cultured with ES-62 (2 μg/mL) for the indicated times and expression of PKCα ((c), Cell Signalling Technology), MyD88 ((e), Abcam) and PKCδ ((e), Cell Signalling Technology) analysed by Western Blotting. In (d), following preincubation for 1 h with inhibitors of proteosomal degradation (10 μM Lactacystin, ENZO Life Sciences; LAC), caveolae/lipid raft trafficking (50 μg/mL Nystatin; NYS) and lysosomal degradation (E64d + pepstatin A both 10 μg/mL, ENZO Life Sciences), sensitised CTMC were cultured with ES-62 (2 μg/mL) for the indicated times and expression of PKCα analysed by Western Blotting. Actin was used as a loading control and ES-62-mediated downregulation of PKCα expression was observed in PDMC, Mucosal-type BMMC, and CTMC in at least 2 independent experiments.