Properdin is secreted by cytokine-activated neutrophils, binds to cells, and promotes the formation of C3/C5 convertases. PMNs were activated as in Figure 2A without the addition of plasma. (A) Supernatants were collected and centrifuged twice at 400g to remove all intact cells. The presence of properdin in these supernatants was measured using a specific enzyme-linked immunosorbent assay described in “Properdin ELISA.” Results are mean properdin concentration plus or minus SD of triplicates from 3 different experiments. (B) Neutrophils, activated as in Figure 2A, without or with incubation with serum one-third, were washed and labeled with a specific antiproperdin mAb. Results are mean plus or minus SD of MFI (n = 4). (C) Cells preactivated or not with TNF/fMLP, as in Figure 2, were incubated in HBSS (no serum, thin line) or in one-third diluted normal (NHS, dark line) or C3-depleted (C3−, dotted line) serum. Cell bound properdin was measured by flow cytometry as in panel B with an antiproperdin mAb, compared with an isotype control (gray peak). Representative experiment from 3 similar ones. (D) TNF/fMLP activated PMNs were incubated with AB-serum (NHS) one-third, with or without 10mM EDTA, or with one-third properdin-depleted serum (P−). They were then labeled with anti-C3d mAb. Results are mean plus or minus SD of MFI (n = 3). (E) Nonactivated neutrophils (2 × 106/mL in HBSS2+) were incubated with increasing concentrations of purified properdin for 20 minutes at 37°C, then either labeled with an antiproperdin mAb (plain line) as in panel B or further incubated with one-third NHS and labeled for cell bound C3d (dotted line). Results are expressed as MFI. (F) Anti-C5b-9 fluorescence histogram of PMNs activated with TNF with or without fMLP and incubated in serum one-third, as in Figure 2A, and labeled with anti-CD5b-9 mAb. Neutrophils incubated in serum with 10mM EDTA represent the negative control (dotted line), whereas the shaded peak was labeled with the IgG1 isotype control. *P < .05. **P < .01. ***P < .001.
