Complement activation is essential for a rapid CpG-induced cytokine production in human whole blood loops. Plasma from whole blood loops was analyzed by CBA for IL-6, TNF, IL-8, IL-1β, IL-12p70, and IL-10 after 1 and 6 h of incubation. Only IL-6, TNF, and IL-8 levels at 6 h are shown in the figure since the other cytokines as well as 1-h samples had low or no cytokine levels. A, Levels of TNF (pg/ml) after stimulation. B, Levels of IL-6 (pg/ml) after stimulation. C, Levels of IL-8 (pg/ml) after stimulation; n = 5–6/group. ***, p < 0.001 and *, p < 0.05 (n.s., not significant) by unpaired t test.

CpG 2006 facilitates AP convertase build-up. QCM-D was used to study the interaction of CpG 2006 with C3b. A, A gold sensor was covered with fibrinogen that was biotinylated in the QCM chamber. Subsequently, NeutrAvidin and biotinylated CpG were added. After that, C3b was injected and incubated. Then the AP convertase was built up using factors B, D, and C3. Each step was followed by washing (except after factors B, D, and C3). Frequency as well as dissipation (not shown) were monitored over time. B, C3b binding reflected by a drop in frequency that corresponds to mass increase on the sensor (black line). Control experiment (gray line) where biotinylated CpG 2006 was omitted in the surface build-up is shown. Normalized frequency was extracted from the third overtone. One representative experiment out of two is shown.

Schematic illustration of the different components involved in CpG-induced complement activation and TLR9 activation and the resulting cytokine secretion and APC activation/maturation that shape the immune response. PS oligos, in this case represented by CpG, activate complement leading to release of anaphylatoxins (C3a and C5a) as well as complement split products that are deposited onto the oligo. Additionally, other molecules such as natural Abs, C1q, as well as properdin bind to the oligo, acting as modulators of complement activation. The activation of complement together with TLR9 stimulation enhances immune activation at the site of inflammation. Both migration and maturation of different cell types are affected. Mo indicates monocytes.

CpG- and GpC-induced complement activation results in increased levels of C3a and C5a. A, Increasing amounts of CpG or GpC were incubated in a circulating whole blood loop system. Each concentration represents the mean ± SD of n = 4 (CpG, n = 2 and GpC, n = 2). Samples were collected after 15 min into EDTA containing tubes and later analyzed by CBA for C3a and C5a levels. B and C, At the highest concentration (60 μg/ml) each individual oligo was investigated separately for complement activation in the whole blood loop system. Compstatin and C5aRA were added to the loops to block C3 and C5aR, respectively. Blood was collected after 1 h and analyzed by CBA. The mean values are shown and data are from three individual experiments.

Addition of CpG or GpC to blood loops results in complement induced CD83 and CD40 expression on monocytes. In the surface-heparinized whole blood loop system the effects of CpG or GpC on monocyte counts and their expression of CD40 or CD83 were monitored by FACS analysis. Compstatin was added to block complement activation and C5aRA to block C5aR. After 1 and 6 h blood was collected into tubes containing EDTA and cells were immediately analyzed with a cell counter and subsequently stained for CD40, CD83, and CD14. A, Monocyte levels after 6 h of incubation. B and C, Levels of CD40 on monocytes (CD14⁺) after 1 h (B) and 6 h (C). D and E, Levels of CD83 on monocytes (CD14⁺) after 1 h (D) and 6 h (E). *, p < 0.05 and **, p < 0.01, unpaired t test (n.s., not significant). Data are from three independent experiments with three different donors (n = 3/bar). Data are presented as fold change compared with a control sample acquired before experiment initiation (mean ± SEM).

Complement inhibition blocks cellular adhesion and uptake of CpG 2006. A, Surface-heparinized 96-well plates were applied in this experiment. CpG 2006-Cy5 was added to 200 μl of whole blood and the plate was placed in 37°C chamber on a rocker and was incubated for 1 h. Compstatin or Abs (10 μg/ml) were added at t = −10 min. At t = 0 min CpG was added. After incubation, blood was collected into EDTA tubes, immediately stained for CD14, and analyzed by FACS. ***, p < 0.001 compared with CpG-Cy5 alone, and **, p < 0.01 compared with CpG-Cy5 plus mouse IgG control (n = 3); unpaired t test, mean ± SD. One representative experiment out of two is shown. B, Confocal images of CpG uptake with or without compstatin. Monocytes were prepared by adhesion of PBMCs to slides with removable chambers. Hirudin plasma was added, and subsequently compstatin and CpG Cy5 were added to the chambers. The cells were incubated for 30 min at 37°C with 5% CO₂. The experiment was terminated by the addition of EDTA. Plasma was saved for later analysis and the chambers were removed. After washing, cells were stained with an anti-CD14 Ab and finally cells were fixated in PFA. Cells were analyzed using confocal microscopy. 4,6′-Diamidino-2-phenylindole (DAPI) (blue) was used to visualize nuclei; anti-CD14 (green) represents monocytes and CpG-Cy5 is visualized in red. A Zeiss 510 Meta confocal microscope was used for imaging, and for acquisition the Zeiss LSM 510 software was applied. The data were acquired using a ×40 oil lens.

P-S CpG 2006 can activate complement both via the alternative and classical pathway in contrast to P-O CpG 2006 and a mixed backbone CpG 2216 that activate complement exclusively via the classical pathway. Hirudin plasma was obtained and plated on a surface-heparinized 96-well plate with oligos added at a final concentration of 8 μM. The plate was rocked at 37°C for 30 min and complement activation was analyzed by measuring levels of C3a and C5a. C5a measurements demonstrated similar results as the C3a data (data not shown). C3a levels in hirudin plasma after incubation with CpG 2006 P-S backbone (A), with CpG 2006 P-O backbone (B), and with CpG 2216 mixed P-S, P-O backbone (C) are shown. Every bar represents triplicates, and significance was tested using an unpaired t test (***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., not significant). Data are shown as means ± SEM. Histograms represents plasma with (filled bars) and without (open bars) EGTA. D, Streptavidin beads coated with biotinylated CpG 2006 and incubated with EDTA plasma were analyzed with FACS for IgM molecules attached to the oligo. For every dilution of plasma, the mean fluorescence intensity (MFI) levels of the control sample (beads without CpG treated with the same amount of plasma) were subtracted from the sample value. Four different donors and four dilutions were assessed. E, Isolated properdin was analyzed for its capacity to bind to CpG 2006. Streptavidin beads coated with biotinylated CpG 2006 were incubated with a dilution series of properdin and analyzed with FACS for properdin CpG 2006 interaction. Values are shown as mean ± SEM. One out of two representative experiments is shown. F, Streptavidin beads coated with biotinylated CpG 2006 and incubated with EDTA plasma were analyzed with FACS for properdin attached to the oligo. For every dilution the MFI levels of the control sample (beads without CpG treated with the same amount of plasma) were subtracted from the sample value. Four donors were assessed. Values are shown as means ± SEM. The experiment was repeated twice and representative data are shown.