Figure 4

Apyrase-sensitive nucleotides derived from dying cells stimulate monocyte chemokinesis. (A) Chemotaxis/chemokinesis of primary human monocytes. HCC1937 cells were treated as in Figure 3A, supernatants were harvested on day 4 after irradiation, and chemotaxis/chemokinesis of primary human monocytes was analyzed by live cell tracking in IBIDI μ-slide chemotaxis 2D chambers. ATP (1 μM) and the FPR agonist WKYMVm (1 μg/ml) served as controls. Trajectory paths of 40 randomly picked cells are shown. Black paths depict cells with net migration upwards, red paths depict cells with net migration downwards. The filled blue circle represents the center of mass after 2 h of migration. (B) Parameters of chemotaxis/chemokinesis. The trajectory paths of 40 randomly picked cells as shown in (A) were analyzed for accumulated distance, euclidean distance (linear distance between start and end position), and the forward migration index in y-direction of the gradient (yFMI = mean of (endpoint in y direction/accumulated distance) of all cells analyzed). Analysis window was set from 10 min to 2 h 10 min (2 h time frame). Bars indicate the median values of 40 cells analyzed, and p-values were calculated by unpaired Student’s t-test. (C) The chemokinesis stimulating factors are sensitive to apyrase treatment. Supernatants of HCC1937 cells irradiated at 20 Gy were collected on day 4 after irradiation and incubated with active or heat-inactivated apyrase (33.3 milliunits apyrase/ml, 50 min at 37°C). Then, they were applied to chemotaxis/chemokinesis assays with primary human monocytes as in (A). Trajectory paths of 40 randomly picked cells are shown. (D) Accumulated distance of the plots shown in (C). Bars depict the median values of 40 cells analyzed, and p-values were calculated by unpaired Student’s t-test.