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Blood, Vol. 106, Issue 5, 1694-1702, September 1, 2005
Heme oxygenase-1 expression inhibits dendritic cell maturation and proinflammatory function but conserves IL-10 expression
Blood Chauveau et al.
106: 1694
Supplemental materials for: Chauveau et al, Vol 106, Issue 5, 1694-1702
Files in this Data Supplement:
- Figure S1. Splenic rat OX62+ DCs analyzed by immunohistology and confocal microscopy (PDF, 158 KB)
- Figure S2. Decrease in HO-1 mRNA levels in matured DCs (PDF, 44.1 KB) -
Rat iDCs were left untreated or incubated with the indicated stimuli for 24 hours. Quantitative RT-PCR analysis results are expressed as transcript accumulation index (TAI), which represents the fold change in mRNA levels in a given sample relative to iDC levels. Results are mean ± SD of 3 experiments. P < .05 for all stimuli versus iDCs.
- Figure S3. Blockade of LPS-induced maturation of DCs by IL-10 (PDF, 124 KB) -
Human iDCs were incubated in the absence or presence of 20 ng/mL IL-10 overnight followed by culture with or without 1 µg/mL of LPS for 24 hours and cytofluorimetric analysis of CD80, CD86, and CD83. Shaded histogram is the signal obtained with the irrelevant mAb IL-10 efficiently blocked maturation of DCs as shown by the inhibited LPS-induced upregulation of CD80, CD86, and CD83. Results are representative of 2 experiments performed in the same conditions.
- Figure S4. Absence of nonspecific signals in human skin sections probed with an irrelevant mAb (PDF, 29.8 KB) -
Cryostat sections of human skin were incubated in the presence or absence of 10 µg/mL of a mouse mAb IgG1 isotype control (identical to the anti–DC-SIGN, DC-LAMP, and HO-1 mAbs used in Figure 4). Tissue sections were then incubated with a biotin-coupled rabbit anti-mouse IgG followed by streptavidin Alexa 568 or a FITC-coupled rabbit anti-mouse IgG. Skin sections analyzed by confocal microscopy do not show nonspecific reactivity.
- Figure S5. Conserved DC viability after porphyrin treatment (PDF, 171 KB) -
(A) Rat iDCs were pulsed for 2 hours with 50 µM cobalt protoporphyrin (CoPP) or tin protoporphyrin (SnPP), washed and cultured for 16 hours, and subsequently incubated with LPS (0.5 µg/mL) for 24 hours. Cells were then analyzed for forward (FSC) and side (SSC) light scatter. Numbers represent percentages of viable cells. No differences were observed between untreated or porphyrin-treated DCs. (B) Human iDCs were pulsed for 2 hours with 50 µM cobalt protoporphyrin (CoPP) or tin protoporphyrin (SnPP), washed and cultured for 16 hours, then incubated with LPS (1 µg/mL) for 24 hours. Cells were subsequently labeled with TO-PRO-3 iodide and analyzed for forward (FSC) and side (SSC) light scatter or by FSC and TO-PRO-3 iodide. Numbers represent percentages of viable cells. No differences were observed between untreated or porphyrin-treated DCs.
- Figure S6. Reduction of ROS in DCs treated with NAC (72.1 KB) -
Rat iDCs were sequentially incubated with 10 mM of NAC for 45 minutes followed by 0.5 µg of LPS for 15 minutes and then 5 µM of the oxidative sensitive dye CM-H2DCFDA for 15 minutes. Cells were then washed and analyzed by cytofluorimetry. A reduction in the fluorescent signal was observed indicating that the signal was specific to ROS and not due to other nonspecific signals. Results are representative of 2 experiments performed in the same conditions.
- Table S1. Phenotype of DCs after porphyrin treatment (PDF, 83 KB) -
(A) Rat iDCs were pulsed for 2 hours with 50 µM cobalt protoporphyrin (CoPP) or tin protoporphyrin (SnPP), washed and cultured for 16 hours, and subsequently incubated with LPS (0.5 µg/mL) for 24 hours. Cells were then analyzed using two-color cytofluorimetry. Values represent the mean of 5 experiments; *P < .01 compared to control LPS-treated cells. (B) Human iDCs were pulsed for 2 hours with 50 µM cobalt protoporphyrin (CoPP) or tin protoporphyrin (SnPP), washed and cultured for 16 hours, and subsequently incubated with LPS (1 µg/mL) for 24 hours. Cells were then analyzed by one-color cytofluorimetry. Values represent the mean of 3 experiments. MFI indicates mean fluorescence intensity; *P < .05 compared to control LPS-treated cells.
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