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Blood, Vol. 107, Issue 12, 4763-4769, June 15, 2006
Adaptive functional differentiation of dendritic cells: integrating the network of extra- and intracellular signals
Blood Luft et al.
107: 4763
Supplemental materials for: Luft et al, Vol 107, Issue 12, 4763-4769
Files in this Data Supplement:
- Figure S1. Independent regulation of CD40L-induced DC migration, expression of co-stimulatory molecules and cytokine secretion by extracellular factors (PDF, 24.7 KB) -
Migration, T-cell activation via co-stimulatory molecules (CD80, CD86) and cytokine secretion are three essential functional characteristics of mature MoDCs. We established activation protocols to investigate whether these functions are independently expressed or linked. A cell line transfected with CD40L (BHK-CD40L) conferred a persistent CD40 signal which induced MoDCs to secrete IL-12p70 and IL-6 (Figure S1A), to express high levels of the T cell co-stimulatory molecules CD80 and CD86 and the maturation marker CD83 (Figure S1B), but to express poor migratory function towards the CCR7 ligand CCL21 (Figure S1C). Addition of the cytokines IFN- and IL-4 increased the secretion of IL-12p70, whereas IL-1 enhanced both IL-6 and IL-12p70 secretion (Figure S1A and 4-6). Migratory capacity and co-stimulatory molecule expression were not significantly altered by these cytokines (Figure S1B, S1C). In contrast, the addition of PGE2 to any of the stimuli combinations tested inhibited IL-12p70 secretion, did not alter IL-6 secretion and enhanced migration as previously reported7,8. Our results demonstrate that these three functional characteristics, i.e. cytokine secretion, co-stimulatory molecule expression and migration, can independently be regulated by extracellular activation stimuli.
MoDCs were generated from CD14+ monocytes within 7 days of culture in the presence of GM-CSF and IL-4. Immature MoDCs were then washed and resuspended in culture medium at a concentration of 1-3×105 cells/ml. BHK-CD40L cells were irradiated and added at a concentration of 1/20 to MoDCs either alone or together with IFN-, IL-1, IL-4 or PGE2. Supernatants and cells were harvested after 36-48 h. A. Secretion of IL-6 and IL-12p70 measured by ELISA (Mean values ± SEM of 5-7 experiments, *p<0.05; **p<0.01 as compared to activation with the BHK-CD40L cell line). B. Mean fluorescence values of CD80, CD83 and CD86 are shown for MoDCs relative to MoDCs activated with the BHK-CD40L cell line (Mean values ± stdev, n=6-14, * p<0.05; **p<0.01 as compared to activation with the BHK-CD40L cell line). C. Migration toward CCL21 (Mean values ± SEM of 3-8 experiments, *p<0.05 as compared to activation with the BHK-CD40L cell line).
- Figure S2. Different sensitivity of migration, expression of co-stimulatory molecules and cytokine secretion to inhibition of persistent p38K and ERK activity (PDF, 14 KB) -
We have previously reported that the removal of the extracellular activation stimuli by washing cultures after 1-3 h preserved expression of migratory function and co-stimulatory molecules, but blocked cytokine secretion2. Using a combination of inhibitors for both, p38K and ERK1/2, we next investigated whether these SRMs also differ in their sensitivity to intracellular signalling blockade. Indeed, cumulative cytokine secretion induced by E. coli could be inhibited if MAPK inhibitors were added as late as 24 h following activation (Figure S2A). In contrast, significant inhibition of migration was only observed within the first 6 h following activation (Figure S2B), and CD80/CD86 expression was likewise not influenced after that time point (Figure S2C).
MoDCs were activated using life E. coli as in Figure 3. p38K inhibitor (p38Ki, 10 µM) and PD98059 (30 µM) were added at the indicated times and cells and supernatants harvested 48 h after activation. A. Inhibition of cumulative cytokine secretion (concentration without inhibitors = 100%) — (concentration with inhibitors added after indicated hours = x%), Means ± SD, n=4 separate donors. B. Inhibition of migration ((migration without inhibitors = 100%) — (migration with inhibitors added after indicated hours = x%)), n=4. C. Inhibition of CD80 and CD86 expression ((mean fluorescence without inhibitors = 100%) — (mean fluorescence with inhibitors added after indicated hours = x%)), Means ± SD, n=3-6 separate donors. Significant differences compared to activation without inhibitors *p<0.05; **p<0.01.
- Figure S3. Adaptive functional differentiation of dendritic cells — integrating the networks of extra- and intracellular players (PDF, 31.4 KB) -
Schematic summary of variables influencing the differentiation of specific DC functional profiles. Specific activation stimuli, such as CD40L or pathogens induce DC activation. Strength and persistence of this extracellular stimulation are essential modulators of the resulting DC functional profile expressed. Extracellular ligand-receptor interactions as well as intracellular signalling pathways (inhibitory and activating) modify the effects of the pathogen, however, the quality of their activity relates to a distinct signal response module (SRM) mediating a specific function. Thus, with regard to DC activation, extra- and intracellular variables may be both, inhibitory and activating (e.g. PGE2, cAMP, ERK1/2), whereas SRMs are either enhanced or blocked. The concert activities of the SRMs constitute the final functional DC profile expressed.
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