Fig 1.
Phosphorylation of Syk in pDCs stimulated by CpG-A or crosslinked with BDCA-2 mAb.
(A) Kinetics of phosphorylation of Syk (Y525/526) in the populations of magnetic bead-sorted pDCs exposed to CpG-A or crosslinked with BDCA-2 mAb was followed by flow cytometry (Phosphoflow). The data show means and SEM of three independent experiments with pDCs from different healthy donors. (B) Kinetics of the total Syk phosphorylation in GEN2.2 cells determined by immunoprecipitation of pTyr followed by Western blotting with Syk Ab. Relative quantity of pSyk was determined by densitometry. Total Syk was used as a loading control. (C) Experimental outline. GEN2.2 cells separated from MS-5 feeder cells and serum-starved overnight in RPMI were exposed or not to Syk inhibitor AB8779 for 1.5 h, and then to CpG-A at 4 μg/ml or to BDCA-2 mAb at 10 μg/ml for 20 min at 4°C. BDCA-2-treated cells were crosslinked with F(ab´)2 for 20 min at 4°C, and followed by analysis of phosphorylation of Syk by Western blotting. (D) Kinetics of phosphorylation of Syk Y352 (pSykY352) and Syk Y525/526 (pSykY525/526) in AB8779-treated or non-treated cells stimulated with CpG-A, BDCA-2 mAb or isotype control (IgG1) was followed by western blot. Total Syk was used as a loading control. Representative result of 3 independent experiments. (E) Quantitative densitometric analysis of phosphorylation of Syk Y525/526 (panel D) in the absence (full columns, [pSyk]) and presence (empty columns, [pSykAB8779]) of AB8779 normalized to the total Syk and expressed as fold increase compared to the control (CpG-A 0 min). ◊, inhibitory index defined by the ratio of pSyk/pSykAB8779 densities. The data show means and SEM, N = 3. *, p ≤0.05; **, p <0.01; two-tailed unpaired Student's t-test.
Fig 2.
Effect of Syk inhibitor AB8779 on production of IFN-α TNF-α and IL-6 in pDCs.
(A) Experimental outline. GEN2.2 cells (B), or primary pDC (C) were incubated with different concentrations of Syk inhibitor AB8779 for 1 hr before stimulation with CpG-A, CpG-B and PMA (N = 3) (B), or CpG-A and R848 (N = 2) (C). After 16 hr culture, IFN-α TNF-α and IL-6 production in GEN2.2 cells (B) or in primary pDCs (C) was determined in cell-free supernatants by ELISA and the results expressed as a multiple of control with the matching concentration of DMSO.
Fig 3.
Subliminal concentrations of Syk inhibitor partially restore IFN-α production in GEN2.2 pDC cell line.
(A) Experimental outline. After separation from MS-5 feeder cells, GEN2.2 cells were incubated with 0.01 μM AB8779 or with a matching concentration of DMSO for 1 hr before exposure ILT7 or BDCA-2 mAb or HCV or HBV particles and stimulation with CpG-A. (B) After 16 hr culture, IFN-α production was determined in GEN2.2 cell-free supernatants by ELISA, and the results were standardized to the quantity of IFN-α produced by GEN2.2 exposed to isotype control Ab or mock-infected culture in the absence of AB8779 (N = 3). (C) IFN-α production determined in GEN2.2 exposed to ILT7 or BDCA-2 mAb or HCV or HBV particles (shown in B) was normalized to IFN-α production in the absence of AB8779 *, p ≤0.05; **, p <0.01.