Fig. 3: Initiation of classical complement activation at the bases of SAP-mediated phagocytosis.

a FITC-labeled AF conidia (1.6 × 10⁶) phagocytosis by freshly isolated human neutrophils (2 × 10⁵) in presence of sera depleted (⁻) of complement components and the effect of human SAP opsonisation after 1 and 30 min. AF conidia (2 × 10⁸) opsonisation with 100 µg of human native SAP. Five percent (left) or 10% (right) of human sera were used. Mean ± SEM of two experiments performed in triplicate and merged. Each spot corresponds to a replicate. Left, NS vs. NS + SAP, **P = 0.009; ***P = 0.002. MBL⁻ S vs. MBL⁻ S + SAP, ***P = 0.002 (two tailed, Mann–Whitney U-test). Right, NS vs. NS + SAP, *P = 0.01; ***P = 0.002. MBL^- S vs. MBL^- S + SAP, *P = 0.04 (two tailed, Mann–Whitney U-test). b FACS analysis of phagocytosis of FITC-labeled AF conidia (5 × 10⁶/200 µl of blood; 20 min) opsonized or not with murine SAP in whole blood of wt, C3-, C1q-, MBL1/2-, and Factor B-deficient mice. Each figure refers to independent experiments performed. Results of C3^−/−, C1q^−/−, and Mbl1/2^−/− are mean ± SEM of one experiment, whereas for Fb^−/− two experiments were merged. Correspondent wt littermates were used as control for C1q-, MBL1/2- (C57BL/6J) and Factor B-deficient mice (C57BL6/NJ). Each spot represents a single mouse. Left, n = 11 wt, n = 10 C3^−/−, n = 8 C1q^−/−. wt vs. C3^−/−, *P = 0.02; wt vs. C1q^−/−, *P = 0.02 (two-sided, unpaired t-test). Middle, n = 5 wt, n = 5 Mbl1/2^−/−. wt vs. Mbl1/2^−/−, *P = 0.01; wt vs. wt + Sap, ***P = 0.004; Mbl1/2^−/− vs. Mbl1/2^−/− + Sap, ***P = 0.002 (two-sided, unpaired t-test). Right, n = 12 wt, n = 14 Fb^−/−. wt vs. wt + Sap, *P = 0.02 (two-sided, unpaired t-test); Fb^−/− vs. Fb^−/− + Sap, **P = 0.005 (two tailed, Mann–Whitney U-test). c FACS analysis of C1q deposition on AF conidia (1 × 10⁷) in presence of plasma from wt (n = 9) or Apcs^−/− (n = 7) mice. Mean ± SEM; *P = 0.05 (two-sided, unpaired t-test). d Effect of blocking of Fcγ receptors in SAP-mediated phagocytosis. Human SAP-pre-opsonized FITC-labeled AF conidia (1.6 × 10⁶) phagocytosis by freshly isolated human neutrophils (2 × 10⁵; 5 min) in presence of NS and anti-CD16, anti-CD32, anti-CD64 or an irrelevant mouse IgG1 (all used at 10 µg/ml). Each spot corresponds to a single healthy donor (n = 4) performed in duplicate. Mean ± SEM. e–g Effect of IgG depletion in SAP-mediated phagocytosis (f) and complement deposition (g). e Western blot analysis for SAP and IgGs in human plasma and after their depletion. 1 µl/lane of plasma-heparin from five healthy donors (A–E) and 10 µg/lane of proteins eluted from the anti-SAP column were loaded. Ponceau red staining was shown. Human SAP (0.1 µg/lane) was used as control. f FACS analysis of phagocytosis by neutrophils (2 × 10⁵) isolated from A–E donors after challenge with FITC-labeled AF conidia (1.6 × 10⁶) opsonized or not with human SAP (100 µg/1 × 10⁹ conidia/ml) in the presence of autologous plasma-heparin (10%) depleted of SAP (SAP^-) and IgGs (IgG^-). g C3 deposition on AF conidia (1 × 10⁷) opsonized or not with human SAP. SAP- and IgG-depleted plasma-heparin (10%) from A–E donors were used. f, g Each spot refers to mean of a duplicate per donor. Mean ± SEM.