Complement activation by neutrophils is not related to apoptosis, serine proteases, oxidants, or a down-regulation of complement control proteins. (A) Anti-C3d histogram of TNF/fMLP-activated PMNs, unwashed (0), washed 3 times, or treated with 1mM phenylmethylsulfonylfluoride for 10 minutes and washed, before the incubation with serum one-third as in Figure 2A. (B) DCFHDA and anti-C3d histograms of a representative experiment (of 3), in which 2 × 10⁶/mL PMNs were preincubated for 1 hour with shaking and in HBSS⁺BSA without Mg, to avoid cell adhesion, without or with 10μM DPI (dotted lines). Mg (1mM) was then added, and cells were activated, incubated in serum, and labeled with anti-C3d as in Figure 2A. To measure the oxidative response, PMNs with or without DPI were treated for 10 minutes with 5μM DCFHDA before cell activation and immediately analyzed, without addition of serum. The shaded peaks represent nonactivated PMNs (0) in the left panel and the control IgG1 isotype in the right panel. (C) Neutrophils, activated as in Figure 2A, were labeled, without addition of plasma, with FITC- or PE-labeled anti-CR1, -DAF, and -MPC mAbs and analyzed by flow cytometry. Results are mean plus or minus SD of MFI normalized with the expression of unactivated PMNs (100%, dotted line). (D) FL1/FL2 dot blots of PMNs activated as in Figure 2A, washed and incubated for 10 minutes in annexin-binding buffer with annexin-FITC and 10 μg/mL propidium iodide, and analyzed by flow cytometry. Apoptotic neutrophils, resulting from a 20-hour incubation at 37°C, were used as positive control. (E) Neutrophil-derived microparticles activate the complement AP. Neutrophils were activated with TNF/fMLP as in Figure 2A and microparticles collected from their supernatant (“Microparticle recovery and labeling”). They were incubated in AB-serum (NHS) one-third, without or with 20mM EDTA or 16.7mM EGTA and 1.6mM Mg and double labeled with anti-CD11b-FITC and biotinylated anti-C3d, followed by streptavidin-PE. The FSC/SSC dot blot shows microparticles (left panel), which are mostly less than or equal to 3 μm.⁸  Fluorescent dot-blots obtained with microparticles in NHS show that most neutrophil-derived, CD11b-positive microparticles are strongly C3d-positive (middle panel, upper right quadrant). The right panel shows the mean plus or minus SD of C3d MFI of CD11b/C3d-positive microparticles released in the various conditions (n = 3).