Figure 1.

The β-chain of C4BP impairs the immunomodulatory activity of its internal CCP6 α-chain domain. (A) SDS-PAGE and Western analysis of the major physiological C4BP isoform (C4BP(β+)) complexed with PS (C4BP(β+)-PS) through its β-chain, and the same C4BP(β+) isoform devoid of PS (C4BP(β+) naked). Both proteins were resolved under reducing (left) and non-reducing (middle) conditions and probed with PK9008 anti-C4BP α-chain antibody. A further Western blot probed with an anti-PS antibody confirmed the absence of PS in the “C4BP(β+) naked” form (right). (B) Human Mo-DCs were incubated throughout their differentiation process with C4BP(β+)-PS, C4BP(β+) naked and C4BP(β-), all at 12 nM (corresponding to 6.0 μg/ml, 5.3 μg/ml, and 5.0 μg/ml, respectively). DC maturation was achieved by LPS treatment. Cells were then collected, washed, and analyzed by flow cytometry for cell surface expression of the activation marker CD83 and the co-stimulatory molecules CD86, CD80 and CD40. MFI, median fluorescence intensity for the different surface markers. (C) The concentrations of IL-12p70, TNF-α and IL-10 were analyzed in the respective cell supernatants by ELISA. iDC, untreated, immature DCs; mDC, untreated, LPS-matured DCs. The results shown are the mean ± SD from 7 independent donors (cell surface markers), or from 4-6 independent donors performed in duplicate (cytokines) (*p < 0.05; **p < 0.01; ****p < 0.0001 compared with mDC). IL-12p70 concentrations induced by C4BP(β+)-PS and C4BP(β+) naked appeared reduced but were not statistically significant compared to that induced by mDC (p = 0.082, and p = 0.053, respectively).