Functional analysis of Ficolin-3 mediated complement activation

Abstract

The recognition molecules of the lectin complement pathway are mannose-binding lectin and Ficolin -1, -2 and -3. Recently deficiency of Ficolin-3 was found to be associated with life threatening infections. Thus, we aimed to develop a functional method based on the ELISA platform for evaluating Ficolin-3 mediated complement activation that could be applicable for research and clinical use. Bovine serum albumin (BSA) was acetylated (acBSA) and chosen as a solid phase ligand for Ficolins in microtiter wells. Binding of Ficolins on acBSA was evaluated, as was functional complement activation assessed by C4, C3 and terminal complement complex (TCC) deposition. Serum Ficolin-3 bound to acBSA in a calcium dependent manner, while only minimal binding of Ficolin-2 and no binding of Ficolin-1 were observed. No binding to normal BSA was seen for any of the Ficolins. Serum C4, C3 and TCC deposition on acBSA were dependent only on Ficolin-3 in appropriate serum dilutions. Deposition of down stream complement components correlated highly significantly with the serum concentration of Ficolin-3 but not with Ficolin-2 in healthy donors. To make the assay robust for clinical use a chemical compound was applied to the samples that inhibited interference from the classical pathway due to the presence of anti-BSA antibodies in some sera. We describe a novel functional method for measuring complement activation mediated by Ficolin-3 in human serum up to the formation of TCC. The assay provides the possibility to diagnose functional and genetic defects of Ficolin-3 and down stream components in the lectin complement pathway.

Figure 1. Binding of recombinant Ficolins to acBSA.

Microtiter plates were coated with acBSA and subsequently incubated with equal concentrations of rFicolin-1 (), rFicolin-2 (), rFicolin-3 () at (A) 30 min at 37°C or (B) 3 h at RT. Binding was detected with monoclonal antibodies directed against the individual proteins. Graphs show mean ± SD (n = 4).

Figure 2. Binding of serum Ficolins and MBL to acBSA.

Microtiter plates were coated with acBSA and subsequently incubated with a NHSP for either (A) 30 min at 37°C or (B) 3 h at RT. Binding of Ficolin-1 (), Ficolin-2 (), Ficolin-3 () or MBL () was subsequently detected with monoclonal antibodies directed against the individual proteins. (C) acBSA incubated with: NHSP (), NHSP with 10 mM EDTA () or NHSP with 10 mM EGTA and 5 mM Mg²⁺ (). Binding was detected with a monoclonal antibody against Ficolin-3. All graphs show mean ± SD (n = 3).

Figure 3. Complement deposition of C4, C3 and TCC on acBSA.

(A–B) Microtiter wells were coated with acBSA or BSA and subsequently incubated with NHSP for 30 min at 37°C. (A) C4 and (B) C3 deposition on acBSA () or BSA () was detected using a polyclonal antibody. (C–D) Microtiter wells were coated with acBSA and subsequently incubated with NHSP (), NHSP with 10 mM EDTA () or NHSP with 10 mM EGTA and 5 mM Mg²⁺ () for 30 min at 37°C. (C) C4 or (D) C3 deposition was detected as described above. (E) Finally, microtiter wells were coated with acBSA () or BSA () and subsequently incubated with NHSP in serial dilution for 45 min at 37°C and TCC-deposition on acBSA was detected using a monoclonal antibody. All graphs show mean ± SD (n = 4).

Figure 4. TCC deposition on acBSA with depleted and deficient sera.

Ficolin-2 or Ficolin-3 was depleted from serum using specific monoclonal antibodies. (A) Depleted serum was applied to SDS-PAGE/Western blotting and probed for Ficolin-2 or Ficolin-3. Lane 1: Ficolin-3 depleted serum. Lane 2: Ficolin-2 depleted serum. Lane 3: Control depleted serum. (B–D) Microtiter plates were coated with acBSA and incubated for 45 min at 37°C with different sera before TCC deposition was detected with a monoclonal antibody. (B) Shows TCC deposition for: NHSP (), Ficolin-2 depleted serum (-♦-), Ficolin-2 depleted serum with addition of 5 µg/ml rFicolin-2 (). (C) Shows TCC deposition for: NHSP (), Ficolin-3 depleted serum (-▪-) and Ficolin-3 depleted serum with addition of 25 µg/ml rFicolin-3 (). (D) Shows TCC deposition for: NHSP (), Ficolin-3 deficient serum −/− (-▴-), Ficolin-3 deficient serum −/− with addition of 25 µg/ml rFicolin-3 (). All graphs show mean ± SD of duplicate wells.

Figure 5. Inhibition of the classical pathway of complement activation with SPS.

(A) Classical pathway activation was obtained by coating microtiter wells with HSA and subsequently incubated with a rabbit-anti-HSA antibody. A NHSP was pre-incubated with or without SPS for 5 min on ice and then applied to the microtiter wells for 30 min (45 min for TCC-detection) at 37°C. Complement activation was detected with biotinylated antibodies. In the absence of SPS: C4 (), C3 () or TCC (); and in the presence of SPS: C4 (), C3 (-•-) or TCC (-□-). (B–D) Different sera pre-incubated on ice with SPS and then incubated for 45 min at 37°C in microtiter plates coated with acBSA before TCC deposition was detected with a monoclonal antibody. (B) Shows TCC deposition in the presence of SPS for: NHSP (), Ficolin-2 depleted serum (- ♦ -), Ficolin-2 depleted serum with addition of 5 µg/ml rFicolin-2 (). (C) Shows TCC deposition in the presence of SPS for: NHSP (), Ficolin-3 depleted serum (-▪-) and Ficolin-3 depleted serum with addition of 25 µg/ml rFicolin-3 (). (D) Shows TCC deposition in the presence of SPS for: NHSP (-▪-), Ficolin-3 deficient serum −/− (-▴-), Ficolin-3 deficient serum −/− with addition of 25 µg/ml rFicolin-3 (). All graphs show mean ± SD of duplicate wells.

Figure 6. TCC deposition on acBSA with Ficolin-3 deficient serum depleted of Ficolin-2 without and with SPS.

Serum pre-incubated on ice (A) without SPS or (B) with SPS and then incubated for 45 min at 37°C on microtiter plates coated with acBSA. The sera analysed was: Ficolin-3 deficient serum −/− (), Ficolin-3 deficient serum −/− depleted of Ficolin-2 (), Ficolin-3 deficient serum −/− depleted of Ficolin-2 with addition of 5 µg/ml rFicolin-2 (). TCC deposition was detected with a monoclonal antibody. Graphs show mean ± SD of duplicate wells.

Figure 7. Correlation of C4 and C3 deposition on acBSA to Ficolin-2 and -3 serum concentrations.

Microtiter wells were coated with acBSA and subsequently incubated with individual serum samples in triplicates for 30 min at 37°C. The relative deposition of C4 and C3 on acBSA from serum from 116 individuals was determined using a NHSP as standard (100%). Ficolin-2 and Ficolin-3 concentrations in serum were determined as described in materials and methods. The graphs show the deposition of C4 as a function of (A) Ficolin-2 or (B) Ficolin-3 serum concentrations and the deposition of C3 as a function of the (C) Ficolin-2 and (D) Ficolin-3 serum concentrations. P-values and Spearman rank (r) were calculated using the GraphPad Prism software. (n = 116).

Figure 8. Complement activation on acBSA using C2, C5 or properdin deficient serum.

Microtiter plates were coated with acBSA and subsequently incubated with NHSP (), C2 deficient serum (), C5 deficient serum () or properdin deficient serum () in serial dilutions for 30 min at 37°C. (A) C4 deposition and (B) C3 deposition on acBSA measured using polyclonal antibodies. (C) TCC deposition on acBSA measured using a monoclonal antibody. All graphs show mean ± SD (n = 3).