C3-Glomerulopathy Autoantibodies Mediate Distinct Effects on Complement C3- and C5-Convertases

Abstract

C3 glomerulopathy (C3G) is a severe kidney disease, which is caused by defective regulation of the alternative complement pathway. Disease pathogenesis is heterogeneous and is caused by both autoimmune and genetic factors. Here we characterized IgG autoantibodies derived from 33 patients with autoimmune C3 glomerulopathy. Serum antibodies from all 33 patients as well as purified IgGs bound to the in vitro assembled C3-convertase. Noteworthy, two groups of antibodies were identified: group 1 with strong (12 patients) and group 2 with weak binding C3-convertase autoantibodies (22 patients). C3Nef, as evaluated in a standard C3Nef assay, was identified in serum from 19 patients, which included patients from group 1 as well as group 2. The C3-convertase binding profile was independent of C3Nef. Group 1 antibodies, but not the group 2 antibodies stabilized the C3-convertase, and protected the enzyme from dissociation by Factor H. Also, only group 1 antibodies induced C3a release. However, both group 1 and group 2 autoantibodies bound to the C5-convertase and induced C5a generation, which was inhibited by monoclonal anti-C5 antibody Eculizumab in vitro. In summary, group 1 antibodies are composed of C3Nef and C5Nef antibodies and likely over-activate the complement system, as seen in hemolytic assays. Group 2 antibodies show predominantly C5Nef like activities and stabilize the C5 but not the C3-convertase. Altogether, these different profiles not only reveal a heterogeneity of the autoimmune forms of C3G (MPGN), they also show that in diagnosis of C3G not all autoimmune forms are identified and thus more vigorous autoantibody testing should be performed.

Keywords: C3 glomerulopathy; C3NeF; C5Nef; Eculizumab; complement.

Figure 1

C3G patients presenting with autoantibodies to the C3-convertase. Thirty-three autoimmune patients with C3G with autoantibodies that bind to the in vitro assembled C3 convertase were identified in the Jena C3G cohort. Based on the binding intensity of autoantibodies to the in vitro assembled C3-convertase patients with high and low binding intensities are identified. Separated in group 1 with high/more C3-convertase binding antibodies and group 2 with weak/less binding antibodies. Each group includes patients who score positive in a C3Nef assay as well as C3Nef negative patients. Cut off was set at arbitrary units of 8.5 (average background binding level of 44 control sera). G1 to G2 and G2 to controls (***p < 0.001).

Figure 2

C3G autoantibodies of group 1 and group 2 influence C3 convertase assembly and dissociation by Factor H. (A) Two group 1 autoantibodies from patients #A and #J with high C3 convertase binding IgGs enhance C3-convertase formation. This enhancing effect is even stronger than the C3 convertase stabilizer and complement activator properdin. In contrast, group 2 autoantibodies from patients #a and# r do not enhance C3 convertase formation. Their effect is comparable to IgGs derived from NHS or from a DEAP-HUS patient with autoantibodies to Factor H or to buffer. (B) The same group1 antibodies when bound to the 3 convertase stabilize the enzyme and enhances resistance to factor H mediated dissociation. C3-convertase assembly in vitro and stabilization by autoantibodies are measured over 45 min by detection of convertase Bb using ELISA. (C) The same experiments as outlined in panel (A) are performed with all 33 patients derived autoantibody fractions and bound IgGs are identified after 45 min. All 12 group 1 antibodies enhance C3-convertase formation and again all group 2 antibodies have no or rather low effects (***p < 0.001). (D) All group 1 autoantibodies stabilize the C3 convertase from dissociation by factor H and all group 2 antibodies lack this activity (***p < 0.001).

Figure 3

Group 1 but not group 2 antibodies enhance C3a generation. Fluid phase C3-convertase is assembled in presence of autoantibodies followed by addition of substrate C3. Following incubation for 45 min, C3a generation is monitored by ELISA. (A) Group 1 autoantibodies increase C3a generation and the effect is dose dependent. In contrast, group 2 antibodies do not enhance C3a formation. (B) When all autoantibodies were evaluated group 1 but not group 2 autoantibodies enhance C3a generation (***p < 0.001). C3a generation by NHS is shown as a stippled line.

Figure 4

C5 Convertase binding. Autoantibody binding to the in vitro assembled C3 convertase was evaluated. In this case, group 1 and also group 2 antibodies bind to the assembled C5-convertase (**p < 0.01). IgGs prepared from NHS show background binding (stippled line). In addition, more group 1 antibodies bind to the C5-convertase as compared to group 2 antibodies (**p < 0.01). The C5-convertase was assembled on an ELISA plate by immobilizing C3b (10 μg/ml) and adding Factor B (1 μg/ml), Factor D (0.5 μg/ml), properdin (1 μg/ml), and C3 (10 μg/ml).

Figure 5

Autoantibodies modulate C5a generation. (A) Representative patient's autoantibodies from group 1 (#A and #J) and group 2 (#a and #r) increase C5a generation significantly when compared to IgGs prepared from NHS compared to control (**p < 0.01, ***p < 0.001). (B) IgGs from all patients except # 607 (group 1), # 328, #2367 (both group 2), enhance C5a generation in a significant manner (*p < 0.05, **p < 0.01, ***p < 0.001). Purified IgGs from each patient was added to complement active NHS at 37°C and the mixture was incubated for 45 min. C5a generation was monitored by ELISA and compared to C5a generation in NHS (stippled line).

Figure 6

Group 1 but not group 2 antibodies affect surface C3b deposition. (A) Two representative group 1 autoantibodies (#J and #A) inhibit C3b deposition and the effect is dose dependent. In contrast, both group 2 antibodies (#a and #r) fail to reduce C3b deposition. C3b deposition was followed with adding increasing amounts of IgGs (10–40 μg) to complement active NHS and after surface deposited C3b was quantitated by ELISA (B) Autoantibodies (each 30 μg) from all patients were tested in the same set up. Again, all group 1, but not group 2 antibodies block C3b surface deposition (***p < 0.001). Purified IgG in (A,B) were added to NHS, and the supplemented serum was added to a LPS-coated microtiter plate. After 1 h C3b deposition was assayed by ELISA. Hemolysis by NHS is marked by a stippled line.

Figure 7

Group 1 but not group 2-antibodies reduce C5b-9 surface deposition and prevent hemolysis. (A) Group 1 autoantibodies (#J and #A) inhibit C5b-9 deposition by about 50% and the effect is dose dependent. In contrast, group 2 antibodies (#a and #r) enhance C5b-9 deposition as compared to NHS derived IgGs. C5-9 deposition was followed upon addition of increasing amounts of IgG fractions (5–20 μg) to NHS. (B) Autoantibody fractions derived from group 1 patients (each 30 μg) inhibit lysis of rabbit erythrocytes (***p < 0.001). Purified IgGs were added to NHS, then this mixture was combined with rabbit erythrocytes. Hemolysis of erythrocytes was assayed by measuring the absorbance. Lysis of erythrocytes in NHS is marked as stipled line.

Figure 8

C5 inhibitor Eculizumab reduces C5a but not C3a generation of convertases with autoantibodies attached. (A) Eculizumab does not affect C3a generation. Fluid phase C3 convertases were assembled in presence of group 1 or group 2 antibodies and C3a release was monitored by ELISA. In the absence of Eculizumab group 1 but not group 2 antibodies induced C3a release (panels 1, 3). C3a generation was not enhanced in group 2 (panels 2, 4). (B) Eculizumab blocks C5a generation of C5-convertases with either group 1 or group 2 antibodies attached (***p < 0.001). Eculizumab was added to NHS together with group 1 or group 2 antibodies (each 30 μg). Following incubation C5a generation was measured by ELISA.