Figure 3.

Increase of cytosolic calcium in skin-derived dendritic cells (DC) upon stimulation with unlabelled C3a and C5a. Cells were cultured for 5 days (a). A proportion was treated with 200 U/ml of tumour necrosis factor-α (TNF-α) (b). After labelling with phycoerythrin-conjugated anti CD1a, DC were loaded with Flou-3 AM, as described in the Materials and methods. C3a (1 µg/ml) (▴) or C5a (1 µg/ml) (•) was then added to suspended cells and [Ca²⁺] was immediately assessed by flow cytometry. C5a was pretreated with a 20-fold molar excess of C17/5 (an anti-C5a monoclonal antibody [mAb]) in a control experiment (▪). Ca-ionophore was used as positive control (▾). C5a led to a transient calcium influx in C5aR⁺ dermal CD1a DC, which could be inhibited by preincubation with anti-C5a mAb (a). After stimulation with 200 U/ml of TNF-α, most of the cells became CD83⁺ and no longer showed calcium influx (b). Arrows refer to the time point when C3a resp. C5a were added to the cell suspension.