Figure 1.
MASP-2 is essential for C3 deposition on S pneumoniae.
Serial dilutions of sera were incubated in microtiter plates coated with S. pneumoniae D39 or N-acetylated BSA (as a control), and C3 deposition determined by ELISA. A. Shows the raw data from one experiment with WT and MASP-2 deficient murine serum (means ±SEM). In B, the experiment was extended to include murine sera deficient in other complement components. Results are duplicates (±SD) and are normalised to the C3 deposition observed in the WT control. C. Time course of C3 activation on S. pneumoniae. 1∶40 diluted murine sera were incubated in microtiter plates coated with S. pneumoniae for the times indicated then C3 deposition assayed. Results are means of duplicates and are representative of three independent experiments. D. FACS analysis of C3 deposition on S. pneumoniae opsonised with MASP-2 −/− serum (black line), WT serum (red) and C4 −/− serum (green). The blue trace shows non-opsonised bacteria. E. Results from 3 independent FACS analyses of C3 deposition (mean fluorescent intensity ±SEM). F. Inhibition of MASP-2 activity with mAb AbD04211 abolished C3b deposition on S. pneumoniae opsonised with C4 deficient serum. (means of triplicates ±SEM; p value from Student's t-test). G. In human serum, MBL deficiency had no effect on C3 deposition, while C4 deficiency had no significant effect on the EC50, but reduces absolute C3b deposition by about 50% (means ±SEM; n = 3 for NHS and MBL −/− serum, 1 for C4 deficient serum). H. Correlation between L-ficolin serum concentration and C3b deposition on S. pneumoniae immobilised on microtiter plates for 47 samples of NHS (solid line shows Fisher transformation of Pearson's correlation coefficient; dashed lines, 95% CI thereof).
Figure 2.
Deposition of C4 breakdown products on S. pneumoniae.
ELSIA plates were coated with S. pneumoniae D39, E. coli or mannan (as positive controls) and incubated with NHS for 1 h at 37°C. Bound C4 was detected using an antibody against C4c (which also detects C4b) (A) or an antibody against C4dg, the last breakdown product of C4 that remains covalently attached to the activating surface (B). C and D: Detection of C4c (C) and C4dg (D) on live S. pneumoniae D39 opsonised with NHS. Green trace shows anti-C4 antibodies; the purple shading, an isotype control Ab.
Figure 3.
Binding of lectin pathway recognition molecules to S. pneumoniae.
Microtiter plates coated with formalin-fixed S. pneumoniae or control substrates were used to capture lectin pathway recognition complexes from WT mouse serum or NHS. Murine MBL-A and MBL-C (A), murine ficolin A (B), murine CL-11 (C) human MBL (D), FCN1 & 3 (E) and FCN2 and CL-K1 (F) were assayed by ELISA, as described in materials and methods. Only ficolin A, CL-11 and FCN2 (L-ficolin) bound to the bacteria. Results are means of duplicates and are representative of three independent experiments.
Figure 4.
MASP-2 deficiency impairs phagocytosis of S. pneumoniae by polymorphonuclear leukocytes.
S. pneumoniae were pre-incubated with murine serum (20% v/v), mixed with freshly isolated human PMN and incubated for 2 hr before being immobilised on microscope slides and stained with eosin Y and azur II (REASTAIN), as described in materials and methods. Bacteria opsonised with WT serum (A) are internalized by PMNs, whereas bacteria opsonised with MASP-2 −/− serum (B) are excluded (arrows). C. Electron micrograph showing pneumococci opsonised with WT serum inside a PMN. D. Samples were removed from the S. pneumoniae/PMN mix at the times indicated and viable bacteria determined. WT serum (crosses) facilitated killing by PMN, whereas MASP-2 −/− serum (open squares) is severely compromised in its ability to opsonise S. pneumoniae. Controls were run in parallel containing non-opsonised bacteria (triangles) and bacteria opsonised with WT serum, but without PMN (circles). Results are means (±SEM) of triplicates.
Figure 5.
Lectin pathway deficiency significantly increases the severity of S. pneumoniae infection.
Lectin pathway deficient mice (open squares) and WT littermates (crosses) were infected intranasally with 1×106 cfu S. pneumoniae D39. C57/BL6 Masp2−/− (A) and C57/BL6 Fcna−/− (B) mice are impaired in their survival of S. pneumoniae infection (Mantel-cox log-rank test). C–F. Viable S. pneumoniae counted in lung homogenates and peripheral blood at the indicated time points after infection. Results in C–E are means (±SEM) of five animals sacrificed at each time point. *p<0.05; **p<0.01 (ANOVA).
Figure 6.
Inhibition of MASP-2 increases the severity of S. pneumoniae infection.
WT C57/BL6 mice were dosed i.p. with 1.0 mg/kg body weight of anti-MASP-2 mAb AbD04211 (open triangles) or an isotype control mAb (crosses) 12 h before being infected with 1×106 cfu S. pneumoniae D39. MASP-2 inhibition significantly worsened survival (A) and there was a corresponding increase in viable S. pneumoniae counted in peripheral blood after infection (B). Treatment with 20 mg/kg body weight ceftriaxone 12 hr before infection and every 12 hr thereafter afforded complete protection from S. pneumoniae in animals injected with AbD04211 (filled triangles in A). Results in B are means (±SEM) of twelve animals from which blood was taken at each time point. *p<0.05 (ANOVA).