Figure 9.
Properdin is critical for AP complement activation on autologous cells but not in the fluid phase. (A) Human red blood cells were lysed in 50% normal human serum in the presence of fH SCR19–20. Addition of an anti-DAF (αDAF) antibody increased lysis. In both cases, lysis was prevented by an anti-properdin (αfP) antibody. ■, fH SCR19–20; ▲, fH SCR19–20 + anti-DAF; ♦, fH SCR19–20 + anti-fP; ▼, fH SCR19–20 + anti-DAF + anti-fP. Assays were performed in triplicates. (B) DAF−/−CD59−/− mouse erythrocytes were lysed by 50% WT mouse serum in the presence of mouse fH SCR19–20 and an anti-Crry antibody (+ fH19–20). Lysis was largely prevented if fP−/− mouse serum was used in the same assay (+ fH19–20). No lysis occurred if EDTA was included in the assays (+ EDTA) or if fH SCR19–20 was omitted (no fH19–20). Assays were performed in duplicates. Data in A and B are normalized to the optical density of osmotically lysed erythrocytes. (C) FACS analysis of C3 deposition on DAF−/−/Crry−/−/C3−/− mouse platelets incubated with serum in vitro. Significant C3 deposition occurred on platelets incubated with WT and C4−/− mouse sera but not fB−/− or fP−/− mouse sera, suggesting properdin-dependent AP complement activation on the platelets. Numbers indicate the percentage of cells in each quadrant. Data in A–C are representative of at least three independent experiments. (D) C3 activation in fH-depleted mouse serum is properdin-independent. Both WT (light gray bars) and fP−/− (dark gray bars) mouse serum incurred time-dependent C3 activation after passing through an anti-fH mAb column to deplete fH (fH dpl) but not after passing through a control mAb column (control). Values for activated C3 were normalized to a reference sample as described in Figure 1, and intact C3 was normalized to C3 in the original serum before affinity column processing. Data are representative of three independent experiments.