Table 1.
The effect of blocking the lectin/ficolin pathways of complement activation on the deposition of C3 fragments and MAC formation on B lymphocytes
| % of control for | Total C3 fragment deposition* | C3b/iC3b fragment deposition* | MAC formation† |
|---|---|---|---|
| NHS (control) | 100 | 100 | 100 |
| + mannose (M) | 98.4 ± 14.7 | 101 ± 30.9 | 92.1 ± 30.4 |
| + GlcNAc (G) | 95.9 ± 7.6 | 99.2 ± 25.3 | 103 ± 20.3 |
| + G + M | 96.7 ± 22.1 | 92.3 ± 30.8 | 98.1 ± 31.0 |
| NHS + anti-FD (control) | 100 | 100 | 100 |
| + M | 102 ± 12.1 | 110 ± 8.42 | 119 ± 32.0 |
| + G | 106 ± 14.8 | 115 ± 8.3 | 102 ± 44.9 |
| + M + G | 101 ± 13.3 | 119 ± 11.0 | 96.2 ± 39.2 |
Mean ± SD for four estimates.
Mean ± SD for three estimates.
Human PBMC from healthy donors were prepared by centrifugation of their citrated blood over Lymphoprep (Nycomed, Oslo, Norway) and their sera were harvested by holding blood collected in anticoagulant-free tubes at room temperature for 1 hr, before centrifugation at 400 g for 5 min. The cells were washed twice in 10 ml VBS (4 mm sodium barbiturate, 145 mm NaCl, pH 7.4, supplemented with 0.8 mm MgCl2) and suspended at a density of 106 cells per ml, in low-absorbing polypropylene tubes (Life Technologies, Paisley, UK) containing 30% v/v autologous serum with or without 5μg/ml rabbit anti-human factor D in VB. Mannose (Man) and/or N-acetylglucosamine (Glc-NAc), both at a final concentration of 50 mm, were added to some of the samples and complement activation was effectuated by incubating the cells at 37° for 30 min The reaction was stopped by adding 2 ml of cold EDTA (20 mm) in phosphate-buffered saline (PBS). After three washes with PBS containing 0.05% NaN3, 0.5% bovine serum albumin (BSA) and 10 mm EDTA (PBS/BSA), the cells were incubated for 2 hr on ice with FITC-conjugated rabbit anti-human C3c or -C3d (Dako A/S, Glostrup, Denmark), or FITC-E11 (murine monoclonal antibody recognizing a C9 neoepitope in MAC)12 in 400 μl PBS/BSA containing 5 mg/ml human immunoglobulin G (Biovitrum, Stockholm, Sweden); 5 μl PE anti-human CD19 (BDBiosciences, Brøndby, Denmark) was included in the mixture to identify the B lymphocytes.
Analyses were performed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) using cell quest software. Data were acquired on 3000 B lymphocytes by combined live gating on the Forward Scatter versus. Side Scatter dot plot and the PE histogram. Complement activation was measured as the difference in mean Fl1 signal (net MFI) between cells incubated in NHS under the various conditions, and cells incubated in VBS alone. Activation in the presence of Man and Glc-NAc and expressed as a percentage of the signals obtained in NHS (± anti-FD) without the sugars is given. The results are given as means ± 1 SD.