Figure 1.
The three pathways of complement activation.
The alternative pathway (AP) is activated when C3 undergoes spontaneous hydrolysis and forms the initial C3 convertase, C3(H2O)Bb in the presence of factor B (fB) and cleavage of bound fB by factor D (fD). The alternative C3-convertase is stabilized by properdin (P). The C3 convertase generates C3b and the subsequent C3-convertases are assembled by C3b and Bb. The lectin pathway (LP) is activated when MBL or other immune lectins bind carbohydrates on pathogens, activating the associated serine proteases (MASPs) which cleave C4 and C2. The first component of the classical pathway (CP) is C1, a complex of C1q and its associated serine proteases C1r and C1s. The CP is initiated by C1q recognition of immune complexes, activating C1r and C1s, again cleaving C4 and C2 generating a C3 convertase characteristic of the CP and the LP (C4bC2a). The C3 convertase cleaves C3 generating C3b and enables assembly of a C5 convertase (C3bC3bBb or C4bC2aC3b), and the C5-convertase product (C5b) initiates assembly of the membrane attack complex (MAC) from C5b-C9. * Initial alternative C3 convertase generated from C3(H2O) and fB.
Table 1.
MAC deposition of human complement-competent and -compromised sera in the three individual complement pathways.
Figure 2.
Al(OH)3 exhausts serum complement.
The figure shows residual complement activity measured by MAC deposition by the three pathways (alternative pathway (AP), lectin pathway (LP), Classical pathway (CP)) in normal human serum (NHS) after incubation with different molarities of Al(OH)3 (37°C, 1 h, in milliQ water) and removal of the Al(OH)3-containing precipitate by centrifugation. 100% represents the complement activity without addition of Al(OH)3. A. Residual complement activity after incubation with vaccine-relevant amounts of Al(OH)3 (10 mM or 100 mM). The figure shows means ± SD of three independent experiments each with double determinations (the SDs are not graphically visible except for LP, 100 mM Al(OH)3). B. Residual complement activity after incubation with low amounts of Al(OH)3. The figure is one out of 3 experiments and shows the means of double determinations. Statistically significant differences were calculated using 2-way ANOVA and Bonferroni posttest (**p<0.01; ***p<0.001). Positive, negative and blank controls provided by the kit manufacturer were analysed in each experiment.
Figure 3.
Time course for Al(OH)3 exhaustion of the three complement pathways.
NHS was incubated with 30(OH)3 at 37°C for the indicated times and MAC deposition by the three pathways (alternative pathway (AP), lectin pathway (LP), classical pathway (CP)) was measured in the supernatant after centrifugation. A. Short term effect of Al(OH)3 on the three complement pathways. B. Long term effects of Al(OH)3 on complement activity. The figures show data from 3 independent experiments with means±SD. P-values for the reduction of complement activity were calculated using 2-way ANOVA and Bonferroni posttest (*p<0.05; **p<0.01; ***p<0.001). Positive, negative and blank controls provided by the kit manufacturer were analysed with each experiment.
Figure 4.
Al(OH)3 depletes C3 from serum.
The concentrations of C3, C4 and different immunoglobulins were determined by nephelometry in NHS treated with Al(OH)3 at 37°C for one hour at the indicated molarities. The Al(OH)3-containing precipitate was removed by centrifugation prior to analysis of the supernatant. A. Concentration of C3 and C4 after treatment with 100 mM Al(OH)3. The figure shows the means ± SDs of two independent experiments. The statistical significance was calculated by 2-way ANOVA and Bonferrroni post-test (** p<0.01). B. Total IgG, IgG1, IgG2, IgA after Al(OH)3 treatment. C. IgM, IgG3, IgG4, C3 and C4 after Al(OH)3 treatment. B and C are from one out of two experiments.
Figure 5.
Time course of anaphylatoxin generation by Al(OH)3 in serum.
The anaphylatoxins were measured in NHS after incubation with 30(OH)3 at 37°C for the indicated times and removal of the Al(OH)3-containing precipitate by centrifugation. High and low positive controls provided by the manufacturer and NHS without addition of Al(OH)3 were included as controls in all experiments. The figure shows means±SD of 2 independent experiments each with double determinations. P-values for the production of anaphylatoxins after 60 minutes were calculated using 2-way ANOVA and Bonferroni posttest (*p<0.05; ***p<0.001).
Figure 6.
Complement components in serum are deposited on Al(OH)3.
NHS was incubated with varying molarities of Al(OH)3 for 1 hour at 37°C, and the protein composition of the precipitates was analysed with monoclonal antibodies directed against the indicated complement components. Figure A shows results for AP components (C3, fB, fH, properdin). Figure B shows results for LP and CP components (mannan-binding lectin (MBL), L-ficolin, C1q, C4) and figure C shows results for C5 and MAC compared with C3. Data are presented as means±SD of 2 independent experiments each with double determinations. A blank control and two mock-antibody controls (mouse anti-β-galactosidase (β-gal) and total mouse IgG) were included in each experiment. 2-way ANOVA and Bonferroni posttest was used to assess the lowest Al(OH)3 concentration, in which the complement deposition was significant versus the control (β-gal) (*p<0.05; **p<0.01; ***p<0.001; ns = not significant).
Figure 7.
Time course for complement components deposition on Al(OH)3.
A) Serum was incubated with 30 mM Al(OH)3 for the indicated times at 37°C and centrifuged. The protein composition of the precipitate was analysed using ELISA with monoclonal antibodies directed against the indicated complement components. Data are means±SD of 2 independent experiments. Controls were as in fig. 6. B) Time-dependent generation of the membrane attack complex (MAC The results are the means of 3 independent experiments. Statistical analysis was performed using 2-way ANOVA and Bonferroni posttest between the specific antibody versus control (β-gal) (A) or between time points (B) (**p<0.01; ***p<0.001; ns = not significant).
Figure 8.
C3 cleavage products are present on the Al(OH)3 precipitate.
The precipitate from serum incubated with 30(OH)3 was solubilised using EDTA and run on a reducing SDS-PAGE gel, blotted onto nitrocellulose and developed using a monoclonal antibody against C3d. First lane is a control sample with serum incubated with pH 5 buffer, second lane is authentic control iC3b and third lane is the solubilised sample from the Al(OH)3 precipitate.
Figure 9.
Al(OH)3-induced complement deposition in complement-compromised sera.
Complement-competent NHS (A), a serum depleted for C3 (B), a factor B-depleted serum (C) and a mannan-binding lectin (MBL)-deficient serum (D) were treated with the indicated amounts of Al(OH)3 for 1 hour at 37°C and the Al(OH)3-containing precipitates were isolated by centrifugation and used for coating on a polystyrene surface and analysed for the complement components C3, C4, MBL, Properdin, factor B and membrane attack complex (MAC), using specific antibodies as previously described. The figure is one out of 2 experiments and shows the mean±SD of double determinations. 2-way ANOVA and Bonferroni posttest was used to assess the lowest Al(OH)3 concentration, in which the complement deposition was significant versus the control (β-gal) (***p<0.001; ns = not significant). Controls included were as described in the legend to fig. 6.
Figure 10.
The effect of inhibitors on complement activation by Al(OH)3.
Membrane attack complex (MAC) deposition was measured using a specific antibody on the precipitate of Al(OH)3-treated serum (30 mM Al(OH)3 for 1 hour at room temperature) with or without prior addition of 10 mM EDTA or 10 mM EGTA +10 mM MgCl2 comparing normal human serum with mannan binding lectin (MBL)-deficient serum, C3-depleted serum and fB-depleted serum. The figure shows means of 3 independent experiments±SDs. 2-way ANOVA and Bonferroni posttest showed a significant difference only for NHS and MBL-deficient serum after addition of EDTA (***p<0,001). Controls included untreated and lipopolysaccharide-treated serum.
Figure 11.
Deposition of complement occurs on human vaccines containing Al(OH)3.
A human serum sample was treated with A) Al(OH)3, B) the Al(OH)3-containing vaccine diTe booster (diphtheria and tetanus vaccine) or C) the Al(OH)3-containing Havrix-vaccine. The Al(OH)3-containing precipitate was isolated by centrifugation and used for coating on a polystyrene surface and analysed for the complement components C3, C4, mannan-binding lectin (MBL), Properdin, factor B and membrane attack complex (MAC) using specific antibodies. The figure shows the mean of double determinations±SD from one of two independent experiments. Statistically significant differences were calculated using 2-way ANOVA and Bonferroni posttest for the specific antibody versus the control (β-gal) (*p<0.05, ***p<0.001).