Autoantibody stabilization of the classical pathway C3 convertase leading to C3 deficiency and Neisserial sepsis: C4 nephritic factor revisited

Abstract

C3 deficiency is a rare disorder that leads to recurrent pyogenic infections. Here we describe a previously healthy 18 y/o Caucasian male with severe meningococcal disease. Total hemolytic activity was zero secondary to an undetectable C3. The C3 gene was normal by sequencing. Mixing the patient's serum with normal human serum led to C3 consumption. An IgG autoantibody in the patient's serum was identified that stabilized the classical pathway C3 and C5 convertases, thus preventing decay of these enzyme complexes. This autoantibody is an example of a C4 nephritic factor, with an additional feature of stabilizing the C5 convertase. Previous patients with C4 nephritic factor had membranoproliferative glomerulonephritis. Two years after presentation, this patient's C3 remains undetectable with no evidence of renal disease. We revisit the role of autoantibodies to classical pathway convertases in disease, review the literature on C4-NeF and comment on its detection in the clinical laboratory.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Figure 1

Western blot of patient’s serum demonstrates C3 degradation fragments. C3 fragments in serum were separated by electrophoresis on a 10% Tris-glycine gel under reducing conditions and transferred to a nitrocellulose membrane. The blot was developed with a polyclonal goat anti-human C3 as described in Methods. Purified C3, C3b, iC3b and C3c and C3dg (10 ng each) served as controls (lanes 1–4). Antigenically-reactive C3 fragments in the patient’s serum were detectable only at low dilutions (1:5 and 1:10-lanes 5 and 6) whereas they were readily detectable at high dilutions of normal human serum (1:500 and 1:1000). The data shown are representative of 3 experiments.

Figure 2

Schematic diagrams of C3 activation and degradation.

Figure 3

Patient’s serum activates C3 in normal human serum (NHS). A. The patient’s serum and NHS were diluted 1:50 in PBS and then mixed together at the indicated ratios and processed as described in Methods. Lanes 1, 9, 10 and 11 are controls (10 ng/lane). At the 1:50 dilution, C3 fragments were not detectable in the patient’s serum (lane 8). Therefore, C3 bands detected by Western blot (lanes 2–7) are derived from C3 in the NHS. Samples (lanes 2–6) were incubated for one hr at 37°C. A representative experiment of 8 independent experiments is shown. B. Undiluted serum from the patient and NHS were mixed together at a 1:1 ratio and incubated overnight at 37°C. Lanes 1, 4, 5, and 6 are controls (10 ng/lane). The patient’s serum utilized in lanes 2 and 3 were obtained ~6 months apart. This experiment was performed one time.

Figure 4

The classical pathway C3 convertase is stabilized in the presence of the patient’s serum. The classical pathway C3 convertase (C4b2a) was established on antibody sensitized sheep erythrocytes (EA) as described in Methods. Patient’s serum, NHS or buffer (natural decay) was added and the convertase allowed to decay at 30° C for the times noted. EDTA-treated guinea pig serum was then added and hemolysis was quantitated following incubation at 37°C for 1 hr. Results are representative of 3 independent experiments. The half-life of the classical pathway convertase in buffer (~25 min as shown by the middle curve) is indefinitely extended in the presence of the patient’s serum (upper curve). The accelerated decay of the convertase upon exposure to NHS (compared to the buffer control) is due to C4b binding protein (C4BP) in the NHS (lower curve).

Figure 5

Stabilizing activity for the classical pathway C3 convertase in the patient’s serum is lost by removal of IgG. Serum IgG was removed by one or two adsorption(s) with Protein G Sepharose. (A) The effect of IgG depleted serum on C3 convertase stability was assessed in an hemolysis assay monitoring convertase decay as described in Methods. Samples were allowed to decay for 30 min at 30°C. Shown is the mean +/−SEM of 3 experiments. Removal of IgG from the patient’s serum (bars 4 and 5) partially abrogated hemolysis mediated by the stabilized convertase whereas there was no effect on IgG removal from NHS (bars 7 and 8). Decay of the convertase in the presence of NHS and IgG depleted patient serum was accelerated compared to the buffer alone control due to the presence of C4BP in the serum. (B) Western blot assessment of the efficacy of IgG removal from sera by Protein G adsorption. The blots were performed as described in Methods and developed using donkey anti-human IgG (1:5,000).

Figure 6

Patient’s purified IgG stabilizes the classical pathway C3 convertase. IgG was isolated from NHS and the patient’s serum using a Protein A column and its effect on C3 convertase stability was assessed as described in Methods. Addition of patient IgG to convertase bearing cells recapitulated the effect produced by intact patient serum (top two curves), whereas IgG from NHS had no impact on the spontaneous decay of the convertase (middle two curves). The accelerated decay of the convertase upon exposure to NHS (compared to the buffer control is due to C4b binding protein (C4BP) in the NHS (lower curve). The data represent two independent experiments (+/− SEM).

Figure 7

IgG purified from the patient’s serum by Protein G stabilizes the classical pathway C5 convertase. Classical pathway C5 convertase sensitized cells were prepared as described in Methods. A. Effect of serum or IgG on the formation of the C5 convertase. C5 convertase formed in the presence of either the patient’s serum or 100 µg of the patient’s IgG was stabilized as evidenced by a T15/T0 ratio of 1.0 (compare bars 4 and 10 to bar 1). Substantial stabilization was observed only in the presence of IgG isolated from Protein G, not Protein A (bar 4 vs bar 8). No stabilization was observed with either NHS or normal IgG. The results shown are the mean +/− SEM of three experiments. B. Effect of whole serum or purified IgG on the stability of preformed C5 convertase. The C5 convertase was preformed on the EA cells as described in Methods. The patient’s IgG isolated by Protein G but not Protein A chromatography stabilized the preformed C5 convertase. Stabilization was not observed using IgG from NHS regardless of the isolation method employed. At the concentration of the patient’s serum used here, the C5 convertase was not stabilized. This is likely due to a concentration effect, as a higher dose of the purified IgG (100 µg) was required for C5 convertase stabilization. The results shown are the mean +/− SEM of three experiments.

Figure 8

Two models of convertase stabilization by the patient’s autoantibody. A. The patient’s IgG binds to and stabilizes the classical pathway C3 convertase. This convertase cleaves C3 to C3b and a fraction of this C3b then forms the C5 convertase. The C5 convertase is also stabilized by the IgG. B. After the C5 convertase is formed, it can still be stabilized by a subset of the autoantibodies.