Active Complement Component 5a (C5a)

Complement Component 5a (C5a) is a component of the complement system which plays a key role in promoting migration and adherence of neutrophils and monocytes to vessel walls. C5a has been proven to be able to induce chemotactic migration of THP-1 cells. Therefore, chemotaxis assay used 24-well microchemotaxis system was undertaken to detect the chemotactic effect of C5a on the human monocytic cell line THP-1. Briefly, THP-1 cells were seeded into the upper chambers (100µL cell suspension, 106 cells/mL in RPMI 1640 with 0.5% FBS) and C5a (50ng/mL and 100ng/mL diluted separately in serum free RPMI 1640) was added in lower chamber with a polycarbonate filter (8µm pore size) used to separate the two compartments. After incubation at 37oC with 5% CO2 for 2h, the filter was removed, then cells in low chamber were observed by inverted microscope at low magnification (×100) and the number of migrated cells were counted at high magnification (×400) randomly (five fields for each filter). Result: C5a is able to induce migration of THP-1 cells. The migrated THP-1 cells in low chamber at low magnification (×100) were shown in Figure 1. Five fields of each chamber were randomly chosen to count the migrated cells at high magnification (×400) and the statistical data was shown in Figure 2.
(A) THP-1 cells were seeded into the upper chambers and 50ng/mL C5a was added in lower chamber, then cells in lower chamber were observed at low magnification (×100) after incubation for 3h;
(B) THP-1 cells were seeded into the upper chambers and 100ng/mL C5a was added in lower chamber, then cells in lower chamber were observed at low magnification (×100) after incubation for 3h;
(C) THP-1 cells were seeded into the upper chambers and serum free RPMI 1640 without C5a was added in lower chamber, then cells in lower chamber were observed at low magnification (×100) after incubation for 3h.
Figure. The chemotactic effect of C5a on THP-1 cells.

Figure. The chemotactic effect of C5a on THP-1 cells

The activity of recombinant human C5a was also measured by its ability to induce N-acetyl-β-D-glucosaminidase release from differentiated U937 human histiocytic lymphoma cells. 3.2*106 differentiated U937 cells were added to the 24-well plate and different concentrations of rhC5a was added to the 24-well plate and incubated at 37 ℃ for 3min. The cells were centrifuged at 400 g for 3min, and the supernatant contained N-acetyl-β-D-glucosaminidase. The enzyme activity of N-acetyl-β-D-glucosaminidase was measured by the substrate of 4-Nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside. The result was shown in figure 1, It was obvious that rhC5a can significantly induce N-acetyl-β-D-glucosaminidase release.