MASP-2 is essential for C3 deposition on <i>S pneumoniae</i>.

<p>Serial dilutions of sera were incubated in microtiter plates coated with <i>S. pneumoniae</i> D39 or N-acetylated BSA (as a control), and C3 deposition determined by ELISA. <b>A.</b> Shows the raw data from one experiment with WT and MASP-2 deficient murine serum (means ±SEM). In <b>B</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. <b>C.</b> Time course of C3 activation on <i>S. pneumoniae</i>. 1∶40 diluted murine sera were incubated in microtiter plates coated with <i>S. pneumoniae</i> for the times indicated then C3 deposition assayed. Results are means of duplicates and are representative of three independent experiments. <b>D.</b> FACS analysis of C3 deposition on <i>S. pneumoniae</i> opsonised with MASP-2 −/− serum (black line), WT serum (red) and C4 −/− serum (green). The blue trace shows non-opsonised bacteria. <b>E.</b> Results from 3 independent FACS analyses of C3 deposition (mean fluorescent intensity ±SEM). <b>F.</b> Inhibition of MASP-2 activity with mAb AbD04211 abolished C3b deposition on <i>S. pneumoniae</i> opsonised with C4 deficient serum. (means of triplicates ±SEM; p value from Student's t-test). <b>G.</b> 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). <b>H.</b> Correlation between L-ficolin serum concentration and C3b deposition on <i>S. pneumoniae</i> immobilised on microtiter plates for 47 samples of NHS (solid line shows Fisher transformation of Pearson's correlation coefficient; dashed lines, 95% CI thereof).