Figure 1
Human malignant hematopoietic cell lines express functional complement anaphylatoxin receptors (C3aR, C5aR) and C5a receptor-like 2 (C5L2). (a) Expression of C3aR1, C5aR1 (also known as CD88) and C5L2 was detected in purified mRNA samples from various myeloid and lymphoid leukemia cell lines by RT-PCR. Samples containing only water instead of cDNA and samples without reverse transcriptase were used in each run as negative controls. Representative agarose gels of the RT-PCR amplicons are shown. (b) Flow cytometric analyses of C3aR1 and C5aR1 expression in the leukemia cell lines, and the representative histograms of C3aR1 and C5aR1 expression are shown. As shown here, the data obtained by FACS is consistent with the RT-PCR results. (c) The effect of recombinant human complement C3a, C5a and C5adesArg proteins on the phosphorylation of p42/44 MAPK and AKTser473 intracellular pathway proteins in both myeloid and lymphoid leukemia cell lines was analyzed by western blotting. These cells (2 × 106 cells per ml) were rendered quiescent for 5 h in RPMI medium containing 0.5% BSA at 37 °C, and afterwards the protein lysates were collected after 5 min of stimulation with C3a (1 μg/ml), C5a (140 ng/ml) or C5adesArg (140 ng/ml) proteins. The cells were also treated with C3a plus a low dose of SDF-1 (10 ng/ml), SDF-1 alone or only serum-free medium. The experiment was carried out twice with similar results, and representative blots are shown.
