Figure 2
Complement C3 and C5 cleavage fragments accelerate migration and adhesiveness of human leukemia cells to fibronectin. (a, c). Chemotaxis of malignant myeloid and lymphoid cells toward different concentrations of C3a (0.5–1 μg/ml), C5a (70–140 ng/ml) and C5adesArg (70–140 ng/ml) through Transwell membranes (8-μm pore size) was assessed. The priming effect of C3a on chemotaxis toward a low dose of SDF-1 was also tested by exposing the cells to C3a (500 ng/ml) plus SDF-1 (10 ng/ml) or C3a (1 μg/ml) plus SDF-1 (10 ng/ml). Before stimulation, cells were rendered quiescent in serum-free medium for 5 h at 37 °C. All leukemia cell lines employed (105 cells per 100 μl per insert) were also evaluated for migration in response to a high dose of SDF-1 (300 ng/ml) or toward RPMI medium containing 0.5% BSA as a positive or negative control, respectively. Three hours post stimulation, loaded inserts were carefully removed, and the migrated cells were afterwards collected and counted by the FACS analysis. Data are extracted from at least triplicate samples in three independent experiments. (b, d) Adhesion of myeloid and lymphoid leukemia cell lines to fibronectin-coated surfaces in response to C3a (0.5–1 μg/ml), C5a (70–140 ng/ml), C5adesArg (70–140 ng/ml), C3a (500 ng/ml) plus a low dose of SDF-1 (10 ng/ml) or C3a (1 μg/ml) plus a low dose of SDF-1 (10 ng/ml). Quiescent cells (3000 cells per 100 μl) were stimulated in medium with 0.5% BSA for 5 min at 37 °C. After the non-adherent cells were removed via three consecutive washes with PBS, the number of adherent cells was directly scored by the microscopic analysis. Cells were also evaluated for adhesion in response to a high dose of SDF-1 (300 ng/ml) or to RPMI medium containing 0.5% BSA as a positive or negative control, respectively. Data were extracted from at least triplicate samples in three independent experiments. In all experiments, the negative control values are normalized to 100%. Data are displayed as means±s.d., with a statistical significance *P<0.05 and **P<0.01 in both migration and adhesion assays between cells exposed to anaphylatoxins and control (unstimulated) cells. (e) The increase in motility of malignant hematopoietic cell lines toward C3a and C5a cleavage fragments is the result of a random chemokinetic response. A checkerboard assay was performed in which C3a (1 μg/ml) and C5a (140 ng/ml) proteins were added at the same time into the upper and the lower Transwell chambers. Data are displayed as means±s.d., with a statistical significance *P<0.05 versus control (unstimulated) cells.
