|
|
Blood, Vol. 113, Issue 23, 5839-5847, June 4, 2009
Ligand of scavenger receptor class A indirectly induces maturation of human blood dendritic cells via production of tumor necrosis factor-
Blood Jin et al.
113: 5839
Supplemental materials for: Jin et al
Files in this Data Supplement:
- Figure S1. Maturation of iMDDCs by fucoidan (JPG, 177 KB)
-
(A) iMDDCs (1 × 106) were treated with medium (iMDDC), fucoidan (50 µg/ml) or LPS (1 µg/ml) for 24 hours. The surface phenotypes of the cells were then assessed by flow cytometry using specific Abs (open histograms) or isotype control Abs (shaded histograms). The surface expression is indicated as the mean fluorescence intensity (MFI) and percentages of positive cells among total cells. (B) iMDDCs were treated as in panel A and then analyzed by confocal differential interference contrast images and fluorescence microscopy with immunostaining using PE-conjugated anti-CD80 or FITC-conjugated anti–HLA-DR Abs. (C) iMDDCs were treated with the indicated concentrations of fucoidan for 24 hours and the expression levels of CD83 were then measured as shown in Fig. 2A. Data represent the mean ± SD of three independent experiments. * p < 0.01 compared to untreated. ** p < 0.001 compared to untreated.
- Figure S2. Effects of fucoidan on IL-12p70 production in untreated or LPS-treated MDDC (JPG, 89.1 KB)
-
(A) After iMDDCs (1 × 106) were treated with or without fucoidan, LPS or TNF-α for 24 hours, the supernatant of the cultures was collected and the concentrations of cytokines were measured by ELISA. (B) iMDDCs were treated with or without fucoidan and/or LPS for 24 hours. In the left panel, the concentrations of cytokines in the supernatants were measured by ELISA. In the right panel, cells were harvested and CD83 expression was then measured as shown in Fig. 2A.
- Figure S3. Role of p38 MAPK in fucoidan-induced maturation of iMDDCs (JPG, 106 KB)
-
iMDDCs were pretreated with MAPK inhibitors (PD98059, 20 µM; SB203580, 1 µM, SP600125, 10 µM) for 1 hour and then cultured with or without LPS or fucoidan. After 24 hours, the expression levels of surface markers (A) and cytokine secretion (B) were determined. * p < 0.05. ** p < 0.01.
- Figure S4. Effects of GSK3 inhibitors on fucoidan- and LPS-induced maturation of iMDDC (JPG, 80.1 KB)
-
iMDDCs were pretreated with LiCl (10 mM), SB216763 (10 µM), or SB415286 (10 µM) for 1 hour and then cultured with or without fucoidan or LPS. After 24 hours, the expression levels of CD83 and cytokine secretion were determined. Data represent the mean ± SD of two independent experiments. * p < 0.05 compared to none.
- Figure S5. Production of IL-12p70 and activation of T cells in coculture of fucoidan-matured MDDCs and CD4+ T cells (JPG, 151 KB)
-
(A) Fucoidan-, LPS-, or TNF–α-matured MDDCs (F-MDDC, L-MDDC, or T-MDDC) were harvested and washed twice with media. These DCs were then pretreated with mitomycin c and mixed with CD4+ T cells (1 × 105) in a 1:25 ratio. Mixtures of the cells were then cultured in the presence or absence of neutralizing Ab against CD40 (5 µg) for an additional 1 day and 5 days to determine the concentrations of IL-12p70 and IFN-γ, respectively. The concentrations of cytokines in the supernatants were determined by ELISA. (B) CD4+ T cells were labeled with CFSE and co-cultured with fucoidan-, LPS-, or TNF-α–matured MDDCs. After 5 days, proliferation of CD4+ T cells was analyzed for CFSE dilution using flow cytometry. * p < 0.05 compared to MDDC. ** p < 0.05 compared to without anti-CD40 neutralizing Abs.
|
|
