Figure 2
Figure 2. DCIR triggering does not affect TLR9-mediated costimulatory molecule expression but inhibits IFN-α production. (A) TLR9 triggering induces BDCA-2 and DCIR down-regulation. Expression of BDCA-2, DCIR and dectin-1 was analyzed directly after cell isolation (left) and after 24 hours of culture with IL-3 and CpG-C (right). Dectin-1 is not expressed under the conditions tested. The numbers indicate Δ MFI (MFI of specific antibody minus MFI of matched isotype control). Data represent 3 independent experiments (top panel). BDCA-2 and DCIR down-regulation in response to TLR9 triggering with CpG-C is statistically significant with a reduction to 14% ± 10% (*P ≤ .001, data represent the mean ± SEM of 3 experiments) for BDCA-2 and to 31% ± 14% for DCIR (*P ≤ .001, data represent the mean ± SEM of 6 experiments; bottom panel). (B) TLR9-induced pDC maturation is not influenced by DCIR triggering. Freshly isolated pDCs were stimulated with 5 μg/mL CpG-C or medium on plates coated with murine anti-DCIR or matching isotype control antibodies. Cells were harvested after 24 hours and stained with anti-CD80, anti-CD86, and anti-CD40 (shaded areas) or isotype control antibodies (dotted lines). The maturation markers CD40 and CD80 were not expressed on resting pDCs, whereas there was some CD86 expression detectable. Expression of all costimulatory molecules was quickly up-regulated in CpG-C stimulated cells irrespective of DCIR triggering. Similar results were obtained for MHC class II (data not shown). The data represent 3 independent experiments. (C) DCIR triggering inhibits TLR9-mediated IFN-α production by human pDCs. PDCs were incubated on plates coated with murine anti-DCIR or isotype control antibodies. Cells were stimulated with CpG-C (5 μg/mL) or left unstimulated (control), and IFN-α production was analyzed after 6 and 8 hours (left, one representative donor). DCIR triggering inhibited IFN-α production by 40% ± 3% compared with isotype control samples (P ≤ .001, right, data represent the mean ± SEM of 3 independent experiments; incubation time 8-24 hours). (D) DCIR triggering inhibits TNF-α but not IL-6 production. Cells were treated as in panel C) and TNF-α and IL-6 secretion were analyzed. DCIR triggering inhibited TNF-α production by 28% ± 5% (P ≤ .002), whereas it did not affect IL-6 production (data represent the mean ± SEM of 3 independent experiments; incubation time 6-24 hours). There was no production of IL-10 or IL-12 detectable (data not shown).

DCIR triggering does not affect TLR9-mediated costimulatory molecule expression but inhibits IFN-α production. (A) TLR9 triggering induces BDCA-2 and DCIR down-regulation. Expression of BDCA-2, DCIR and dectin-1 was analyzed directly after cell isolation (left) and after 24 hours of culture with IL-3 and CpG-C (right). Dectin-1 is not expressed under the conditions tested. The numbers indicate Δ MFI (MFI of specific antibody minus MFI of matched isotype control). Data represent 3 independent experiments (top panel). BDCA-2 and DCIR down-regulation in response to TLR9 triggering with CpG-C is statistically significant with a reduction to 14% ± 10% (*P ≤ .001, data represent the mean ± SEM of 3 experiments) for BDCA-2 and to 31% ± 14% for DCIR (*P ≤ .001, data represent the mean ± SEM of 6 experiments; bottom panel). (B) TLR9-induced pDC maturation is not influenced by DCIR triggering. Freshly isolated pDCs were stimulated with 5 μg/mL CpG-C or medium on plates coated with murine anti-DCIR or matching isotype control antibodies. Cells were harvested after 24 hours and stained with anti-CD80, anti-CD86, and anti-CD40 (shaded areas) or isotype control antibodies (dotted lines). The maturation markers CD40 and CD80 were not expressed on resting pDCs, whereas there was some CD86 expression detectable. Expression of all costimulatory molecules was quickly up-regulated in CpG-C stimulated cells irrespective of DCIR triggering. Similar results were obtained for MHC class II (data not shown). The data represent 3 independent experiments. (C) DCIR triggering inhibits TLR9-mediated IFN-α production by human pDCs. PDCs were incubated on plates coated with murine anti-DCIR or isotype control antibodies. Cells were stimulated with CpG-C (5 μg/mL) or left unstimulated (control), and IFN-α production was analyzed after 6 and 8 hours (left, one representative donor). DCIR triggering inhibited IFN-α production by 40% ± 3% compared with isotype control samples (P ≤ .001, right, data represent the mean ± SEM of 3 independent experiments; incubation time 8-24 hours). (D) DCIR triggering inhibits TNF-α but not IL-6 production. Cells were treated as in panel C) and TNF-α and IL-6 secretion were analyzed. DCIR triggering inhibited TNF-α production by 28% ± 5% (P ≤ .002), whereas it did not affect IL-6 production (data represent the mean ± SEM of 3 independent experiments; incubation time 6-24 hours). There was no production of IL-10 or IL-12 detectable (data not shown).

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