A Monoclonal Antibody That Provides a Model for C3 Nephritic Factors

Abstract

Complement is a major innate defense system that protects the intravascular space from microbial invasion. Complement activation results in the assembly of C3 convertases, serine proteases that cleave complement protein C3, generating bioactive fragments C3a and C3b. The complement response is rapid and robust, largely due to a positive feedback regulatory loop mediated by alternative pathway (AP) C3 convertase. C3 nephritic factors (C3NEFs) are autoantibodies that stabilize AP convertase, resulting in uncontrolled C3 cleavage, which, in principle, can promote critical tissue injury similar to that seen in certain renal conditions. Investigations of C3NEFs are hampered by a challenging issue: each C3NEF is derived from a different donor source, and there is no method to compare one C3NEF to another. We have identified a widely available mouse anti-C3 mAb that, similar to many C3NEFs, can stabilize functional AP convertase in a form resistant to decay acceleration by multiple complement regulators. The antibody requires the presence of properdin to confer convertase stability, and hampers the activity of Salp20, a tic salivary protein that accelerates convertase dissociation by displacing properdin from the convertase complex. This mAb can serve as an urgently needed standard for the investigation of C3NEFs. This study also provides novel insights into the dynamics of AP convertase.

Introduction

Complement (C) is a major innate defense system that protects the intravascular space from microbial invasion.¹ C activation results in the assembly of C3 convertases, serine proteases that cleave the complement protein C3, generating the bioactive fragments C3a and C3b, leading to downstream events, including target clearance and/or lysis, and the activation and recruitment of immune cell types.^1–4 The C response is rapid and robust, largely because of a positive feedback regulatory loop mediated by the alternative pathway (AP) C3 convertase.⁵

AP C3 convertase assembly begins with the covalent attachment of nascent C3b to the target surface.¹ This is followed by the association of C3b with factor B (FB), the cleavage of C3bB by factor D (FD) at a single FB site, and the association of C3bBb with properdin (P). C3b serves to direct complement activity and FB cleavage activates the Bb protease domain. Properdin stabilizes the C3bBbP complex (T1/2 = 10–20 minutes).^6,7 Once dissociation occurs, catalytic activity of the Bb subunit is lost. AP convertase activity is controlled by regulatory proteins that protect the host cells and tissues from detrimental C activity. Several host regulators deactivate convertases by promoting the dissociation of Bb from C3b (decay acceleration), whereas others serve as cofactors for inactivation of C3b by serine protease factor I (FI).⁸ The failure of these proteins to control AP convertase activity can lead to critical tissue injury.

C3 nephritic factors (C3NEFs) are a highly heterogeneous group of autoantibodies that stabilize AP convertase.^9–11 They are observed in rare renal conditions, including 40%–50% of patients with C3 glomerulonephritis (previously classified as membranoproliferative glomerulonephritis types I and III) and 70%–80% with dense deposit disease (previously membranoproliferative glomerulonephritis type II).¹² Most C3NEFs recognize C3 neoepitopes expressed in AP C3 convertase, stabilizing the active complex and conferring resistance to decay acceleration mediated by factor H (FH), and in some cases by decay-accelerating factor (DAF) and/or complement receptor 1 (CR1).^13,14 These activities can result in uncontrolled C3 cleavage, which, in principle, promotes critical tissue injury similar to that observed in rare renal conditions.

We identified a widely available mouse anti-C3 mAb that, similar to many C3NEFs, can stabilize functional AP convertase in an FH- and DAF-resistant state. This mAb can serve as an urgently needed standard for the investigation of C3NEFs. This study also provides novel insights into the dynamics of AP convertase.

Materials and Methods

Proteins and reagents

Anti-C3 mAb clone 7C10 (also denoted as HAV-003-05) was purchased from Cedarlane. It was developed by Statens Serum Institut (Copenhagen, Denmark). The C3d specificity of 7C10 was reported by the manufacturer and confirmed by comparing its reactivity with C3, C3b, iC3b, C3c, and C3d by western blotting (not shown). Other anti-C3 antibodies were obtained from the Antibodyshop and Quidel. Recombinant CD55/DAF was from R&D Systems (Catalog No. 2009-CD-050). Salp20 was a gift from Dr. Katherine Tyson and Dr. Aravinda de Silva (University of North Carolina, Chapel Hill, USA). Soluble CR1 (sCR1) was a gift from Dr. John Atkinson (Washington University School of Medicine, St. Louis, MO, USA). All other complement proteins were obtained from CompTech (Tyler, Texas, USA). Sheep blood cells and guinea pig serum were obtained from Colorado Serum Company.

Buffers

DGVB²⁺ buffer: 1 mM MgCl₂, 0.15 mM CaCl₂, 71 mM NaCl, 0.1% (w/v) gelatin, 2.5% (w/v) dextrose, and 2.47 mM sodium 5′,5″-diethyl barbiturate (pH 7.35); Mg²⁺ EGTA buffer: 10 mM Na₂EGTA, 7 mM MgCl₂, 59 mM NaCl, 0.083% (w/v) gelatin, 2.075% (w/v) dextrose, and 2.05 mM sodium 5′,5″-diethyl barbiturate (pH 7.3–7.6); 10 mM EDTA buffer: 10 mM Na₂EDTA, 128 mM NaCl, 0.1% (w/v) gelatin, and 4.45 mM sodium 5′,5″-diethyl barbiturate (pH 7.35); 40 mM EDTA buffer: 40 mM Na₂EDTA, 85 mM NaCl, 0.1% (w/v) gelatin, and 2.96 mM sodium 5′,5″-diethyl barbiturate (pH 7.35).

Preparation of C3b-opsonized sheep cells

Cells were prepared by first assembling classical pathway C3 convertases on sheep erythrocytes and then using them to coat the cell surface with C3b.¹⁵ Ab-sensitized sheep erythrocytes (5 mL, 5 × 10⁸/mL) obtained from CompTech were washed twice and resuspended in 5 mL of DGVB²⁺ buffer, mixed with 37.5 μg of human C1 in 5 mL of DGVB²⁺, and incubated for 15 minutes at 30°C. The resulting cells were washed twice, resuspended in 5 mL of DGVB²⁺, mixed with 50 μg of human C4 suspended in 5 mL of DGVB²⁺, and incubated for 15 minutes at 30°C.

These cells were washed twice, suspended in 5 mL of DGVB²⁺, mixed with 250 μg of human C3 and 5 μg of human C2 suspended in 5 mL of DGVB²⁺, and incubated for 30 minutes at 30°C. The resulting cells were washed and resuspended in 5 mL of 10 mM EDTA buffer and incubated at 37°C for 2 hours to allow for dissociation of the active classical pathway convertases. The resulting C3b-coated cells were washed twice in 5 mL 10 mM EDTA buffer, twice in 5 mL of 10 mM Mg²⁺ EGTA buffer, and resuspended in 10 mM Mg²⁺ EGTA buffer to a final concentration of 1 × 10⁸/mL. They were stored at 4°C and used within 1 week.

Effects of anti-C3 mAb on the activity of cell-bound C3bBbP complexes

C3b-opsonized sheep erythrocytes (100 μL), 50 μL of purified FD (5 ng), 50 μL of properdin (45 ng), and 50 μL of FB (2 ng) were mixed in Mg²⁺ EGTA buffer and incubated at 30°C for 30 minutes. Cells were chilled to 4°C and treated for 5 minutes with 150 μL 40 mM EDTA buffer containing 1 μg of mouse anti-human C3 mAb and/or a complement regulator (FH, DAF, sCR1). Samples were then incubated for various times at 30°C. Functional convertases were then quantified by adding 150 μL of a 1:20 dilution of guinea pig serum in 40 mM EDTA buffer (serum-EDTA), followed by incubation at 37°C for 60 minutes.

Additional samples included cell lysis controls in which cells were treated with 450 μL of distilled water alone, and a negative control in which cells were treated with 450 μL of DGVB²⁺ buffer alone. All samples were then centrifuged, and the OD₄₁₄ of the supernatant was determined. In some experiments, the hemolytic activity was expressed as the percentage of cell lysis. In most experiments, hemolytic activity levels were expressed as the Z metric. Z is defined as the average number of lytic sites formed per red blood cell and is calculated using the expression Z = −ln (1 − y), where y is the proportion of lysed cells.¹⁶

Effect of 7C10 on the activity of cell-bound C3bBb complexes

The general protocol (aforementioned) was followed, except that properdin was excluded, and additional FB was employed (10 ng total).

Results

C3NEFs are a highly heterogeneous group of antibodies, the properties of which differ from one donor source to another. To date, there has been no standard method to compare one C3NEF to another. We posit that a widely available mAb that shares many of the properties of C3NEFs might resolve this issue. To this end, we screened anti-C3 mAbs for their effect on AP convertase stability. Convertases were assembled on C3b-coated sheep red blood cells using purified FB, FD, and properdin (P). Cells bearing convertases were then incubated for up to 2 hours with an anti-C3 mAb in EDTA buffer that blocked further convertase assembly. Hemolysis was assayed after the addition of guinea pig serum-EDTA mix (see Materials and Methods section).

During incubation, the preassembled complexes undergo spontaneous dissociation, resulting in reduced cell lysis. In contrast, in several cases, the inclusion of anti-C3 mAb stabilized convertases, and cell lysis remained near 100% (Fig. 1A). 7C10, an antibody that recognizes the C3d fragment, stabilized over 75% of the potential lytic events over a 2-hour period (Fig. 1B). Previously, we and others have described C3d-specific mAbs that stabilize C3bBbP, but none conferred FH resistance.^17,18 In contrast, 7C10 conferred a high level of FH resistance (Fig. 1C). Low or no FH resistance was afforded by several other anti-C3d specific mAbs (not shown).

FIG. 1.

Identification of a C3NEF-like mAb. C3bBbP were assembled from FB, P, and FD on C3b-opsonized sheep red blood in Mg²⁺ EGTA buffer. (A) C3bBbP-bound cells were treated with various anti-C3 mAb in 40 mM EDTA buffer and incubated for 0 or 2 hours at 30°C before treatment with serum-EDTA. (B) C3bBbP-bound cells were treated with mAb 7C10 in 40 mM EDTA buffer and incubated for 0 or 2 hours at 30°C before addition of serum-EDTA. Data compiled from three experiments. (C) C3bBbP-bound cells were treated with FH in 40 mM EDTA buffer for 5 minutes at 4°C before addition of serum-EDTA. Data compiled from three experiments. Z = average lytic sites per cell. C3NEF, C3 nephritic factor; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; FB, factor B; FD, factor D; FH, factor H.

Properdin is the only positive regulator of AP. It increases C3bBb half-life 5- to 10-fold by stabilizing the C3b:Bb interface.^6,7 Two general classes of C3NeF have been delineated, one class requiring properdin for activity and the other active in its absence.^{13,14,19–21} In the absence of properdin, 7C10 conferred FH resistance (Fig. 2A), but it did not confer stability; nearly all hemolytic activity was lost within 15 minutes (Fig. 2B). These experiments suggest that 7C10 may lend stability to convertase by supporting the interaction between properdin and C3bBb. To explore that possibility, we examined the effects of 7C10 on Salp20, an Ixodes scapularis salivary protein that inhibits the AP by binding properdin and displacing it from the convertase complex.^22,23 As shown in Figure 3A, 7C10 conferred resistance to the effects of Salp20 on the preassembled C3bBbP. Additional experiments established that 7C10 conferred resistance to two other regulatory proteins, DAF and CR1 (Fig. 3B, C).

FIG. 2.

Anti-C3 mAb 7C10 confers FH resistance but is not stable to convertase preformed without properdin. C3bBb was assembled from FB and FD on C3b-opsonized sheep red blood cells in Mg²⁺ EGTA buffer. (A) C3bBb-bound cells were treated with FH in 40 mM EDTA buffer for 5 minutes at 4°C, before addition of serum-EDTA. (B) C3bBb-bound cells were treated with mAb 7C10 in 40 mM EDTA buffer for 5 minutes at 0°C and incubated for 0, 15, or 30 minutes at 30°C before the addition of serum-EDTA. Data from each panel were compiled from three experiments. Z = average number of lytic sites per cell.

FIG. 3.

Anti-C3 mAb 7C10 confers resistance to Salp20, DAF and sCR1. (A–C) C3bBbP was assembled from FB, P, and FD on C3b-opsonized sheep red blood in Mg²⁺ EGTA buffer. C3bBbP-bound cells were treated with sCR1, recombinant DAF, or Salp20, with or without anti-C3 mAb 7C10, in 40 mM EDTA buffer for 5 minutes at 4°C before addition of serum-EDTA. The data in panels A and B are compiled from three experiments. The data in panel C are compiled from five experiments. Z = average number of lytic sites per cell. DAF, decay-accelerating factor; sCR1, soluble CR1.

Discussion

C3NEFs comprise a functionally diverse group of autoantibodies. In this study, we describe the properties of 7C10, a mouse monoclonal antibody that mimics properties attributed to many C3NeFs:7C10 stabilizes AP convertases in a form resistant to FH. The effect on stability requires properdin, whereas FH resistance does not. 7C10 also confers resistance to DAF and partial resistance to sCR1. In many experiments presented in this study, 7C10 enhanced hemolytic activity, even in the absence of properdin (see Fig. 2). Several groups have reported that a subset of C3NEFs elevates C3 convertase and/or C5 convertase catalytic activities.^14,24,25

C3NEFs are often accompanied by rare conditions characterized by the accumulation of C3 in the renal tissue.¹² However, their role in disease is unclear, in part due to reports of C3NEFs in healthy individuals.^26–28 Although they are known to bind and stabilize AP convertases, they have been identified using a variety of assays that may differ in sensitivity and clinical significance. Autoantibodies that do not confer FH resistance may have clinical consequences.¹⁴

The capacity of 7C10 and other C3d-specific mAbs to inhibit the effects of FH, and possibly DAF and CR1, could be largely due to steric hindrance because these regulators bind to a common region adjacent to the C3b thioester-containing domain (TED) domain.^29,30 In contrast, the capacity of 7C10 to confer properdin-dependent convertase stability and disrupt the displacement of properdin by Salp20 strongly suggests that 7C10 supports the interaction of properdin with C3bBb. The known convertase structure (Fig. 4) strongly suggests that this effect is indirect one.³¹ The TED domain is located ∼75 Angstroms from the C3b:Bb interface where C3bBb is held together by properdin that binds to the C3 carboxy-terminus at the C3b:Bb interface.^32,33

FIG. 4.

3D structure of C3bBb. The C3b:Bb interface, stabilized by Mg(2+), is distal to the TED domain at the 7C10 recognition site. Bb is blue, the C3b carboxy terminal domain is salmon, the TED (C3d) domain is green, and the thioester reactive moiety is labeled light blue. Mg(2+) is shown in red. Structure generated from PDB ID:2WIN with PyMOL Molecular Graphics System, Version 2.5.1 Schrödinger, LLC. TED, thioester-containing domain.

Although X-ray crystallographic analyses of free C3b and C3b complexes have established a static arrangement of many C3b domains, little is known about the dynamics of these domains. Electron microscopy studies have shown that C3 and its products demonstrate a remarkable capacity for domain rearrangement and flexibility, which is most evident in the TED domain.^34–37 7C10 and other antibodies that bind to the C3b TED domain may support the P-stabilized Bb:C3b interface by modulating the repertoire of convertase conformations that impacts that region.

Investigations of the C3NEF mechanism(s) of action are confronted by a challenging situation: each C3NEF is derived from a different donor source, and there is no available standard of comparison. Moreover, structural studies are hampered by the fact that C3NEFs occur in heterogeneous antibody populations. The 7C10 mAb is offered in purified form by multiple commercial sources and, therefore, has the potential to provide an urgently needed standard for C3NEF studies. It can also be used to model the effects of C3NEFs in detailed structure and function studies.

Authors' Contributions

Conceptualization, methodology, data curation, writing—original draft, funding acquisition, and supervision by D.E.H. Methodology, investigation, writing—reviewing and editing, and data curation by L.M.M.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was funded by NIH grants R21AI137223 (D.E.H.) and R01AI051436 (D.E.H.).