Results were depicted as differences of the means between LPS-treated and untreated cells. As shown in Figure 2a, rapid cell swelling was observed in WT DCs 30 min after the addition of LPS. Thereafter, the cell size of LPS-treated R428 supplier WT DCs remained on a high level up to 120 min and decreased after 180 and 240 min, respectively. By contrast, in KCa3.1-deficient DCs, only a very moderate swelling was observed between 30 and 60 min after addition of LPS. These results suggest that KCa3.1 is important for DC swelling in an initial-to-middle phase between 30 and 120 min upon LPS treatment. In migrating cells elevated free cytosolic

Ca2+ concentrations were observed [19]. Moreover, treatment of DCs with LPS or supernatants of Escherichia coli was followed by a rapid increase in [Ca2+]i [7, 20]. Hence, changes in [Ca2+]i after stimulation with LPS were monitored in WT and KCa3.1-deficient BMDCs using the Ca2+-sensitive dye fluo-3 AM. Results were depicted as differences of the means of fluorescence intensities Rapamycin between LPS-treated and untreated cells. As shown in Figure 2b, a gradual increase in the free cytosolic Ca2+ concentration was observed in WT DCs starting at 30–90 min after the addition of LPS reaching a plateau

at 180–240 min. By contrast, the increase in [Ca2+]i was much lower in LPS-treated KCa3.1−/− DCs indicating that the LPS-induced changes in [Ca2+]i depend on KCa3.1 activity and thereby the channel might be important for the LPS-induced migration in DCs as well. In order to directly analyze the role of KCa3.1 for the Myosin LPS-induced DC migration, transwell assays were performed with

WT and KCa3.1-deficient BMDCs (Fig. 2c). The activity of DCs to migrate toward a CCL21 gradient was depicted as the migration rate to CCL21 divided by the migration rate to medium alone (chemotactic index). According to the results shown in Figure 2c, WT DCs kept in medium did not migrate in a CCL21-directed manner (chemotactic index: 1.1), whereas treatment of WT DCs with LPS for 4 hr caused an increase in CCL21-directed migration (chemotactic index: 2.1). By contrast, the migratory activity of untreated KCa3.1-deficient DCs was comparatively high (chemotactic index: 1.9). However, after treatment with LPS KCa3.1−/− DCs migrated to a less extend (chemotactic index: 1.4) when compared to WT DCs suggesting that the KCa3.1 channel is involved in LPS-induced DC migration. Migration of cells in response to inflammatory stimuli is an essential component in host defense. In neutrophils stimulated with the chemoattractive peptide fMLP an increased cell volume through activation of sodium/proton antiport causing intracellular accumulation of ions and subsequent water influx is a prerequisite for cell migration [12, 21]. Moreover, in DCs it has been demonstrated previously that LPS induces cell swelling by transient activation of the Na+/H+ exchanger [13]. Accordingly, we here show that treatment with LPS rapidly causes cell swelling (Fig. 1a) and migration (Fig.