Figure 4

Sublytic perforin induces a transient Ca²⁺ influx. (A) – (D), and (F), DC were loaded with Fura-2 for 15 minutes prior to the incubation with perforin in a concentration of 2.5 kU/ml. At indicated time points fluorescent images (Excitation 340 nm; Emission 525 nm) were recorded (A). The excitation ratio 340/380 nm was monitored over the whole experimental period (B). Some cultures were pretreated with TMB-8 (10 μM) and dantrolene (25 μM) to block intracellular Ca²⁺ mobilization (C). (D), DC were equilibrated in a low Ca²⁺ (1.3 μM) buffer before a first exposure to perforin, after washout and buffer exchange to normal Ca²⁺ conditions (1.3 mM) the cells were again treated with perforin. ATP and ionomycin were used as positive controls for Ca²⁺ mobilization from intracellular or extracellular sources. One representative measurement of more than six independent experiments is presented. (E), L. innocua-challenged DC were incubated for 3 hours with granulysin in presence or absence of 2.5 kU/ml perforin or 1 μM ionomycin at 37°C or were incubated with ionomycin alone. After the incubation, the cells were lysed in ice-cold water and Listeria viability was calculated from bacterial growth curves. Mean values and SD of three independent experiments are presented. (F), to demonstrated typical Ca²⁺ responses to ionomycin, DC were pretreated with Fura-2 for 15 minutes prior to the incubation with ionomycin (1 μM). One representative measurement out of four independent experiments is presented.