Alternative Pathway Dysregulation and the Conundrum of Complement Activation by IgG4 Immune Complexes in Membranous Nephropathy

Heparin induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing IgG antibodies bound to multivalent complexes of platelet factor 4 (PF4) and heparin (H). HIT ULICs activate cellular FcγIIA receptors that initiate diverse cellular effector functions including neutrophil degranulation and monocyte expression of tissue factor (TF). Previous studies have shown that HIT ULICs also potently activate complement through the classical pathway (Cines et al., 1980). Whether complement activation contributes to FcγRIIA-dependent prothrombotic pathways has not been addressed in detail. In studies that follow, we describe: 1) robust complement activation by HIT ULICs in plasma and whole blood (WB), 2) cell-surface deposition of complement and IgG triggered by HIT ULICs, 3) complement-dependent neutrophil degranulation and monocyte TF expression, 4) efficacy of proximal, but not terminal, pathway inhibition in regulating monocyte TF expression, and 5) deposition of complement in thrombi formed in "HIT mice" that generate ULICs containing KKO, a HIT-like monoclonal antibody (Arepally et al., 2000). Consistent with prior studies showing involvement of the classical pathway in HIT (Cines et al., 1980), we observed that binding of C1q induced marked enlargement of HIT ULICs in buffer assessed by dynamic light scattering as well as in plasma using confocal microscopy (data not shown). To assess complement activation by HIT ULICs, we incubated WB and plasma with PF4 (25 µg/mL) ± heparin (1 U/mL) in the presence of KKO (or isotype, "ISO"; 50 µg/mL) or HIT IgG (or control IgG, "CON"; 500 µg/mL) and measured C3c with a capture immunoassay as previously described (Khandelwal et al., 2018). KKO (Figure 1A) or HIT ULICs (n=3; HIT1-3, Figure 1B), showed robust generation of C3c in the presence of PF4/heparin, but not antigens alone or with control IgG (ISO/CON). Complement activation by HIT ULICs leads to downstream generation of C5a and formation of sC5b-9 (data not shown). Pre-incubation of plasma or WB with a variety of classical pathway inhibitors, including a C1r inhibitor derived from Borrelia burgdorferi (BBK 32), C1 esterase inhibitor (Berinert, CSL Behring) and anti-C1q antibody (α-C1q Ab; Annexon Biosciences) inhibited C3c generation by KKO ULICs (p <0.001), whereas inhibitors of the alternative pathway (anti-properdin antibody) or C5 inhibitor (α-C5 Ab; Eculizumab, Alexion Pharmaceuticals) did not (data not shown). Incubation of WB with KKO or HIT ULICs, but not ISO or CON IgG, markedly increased deposition of C3 and IgG on neutrophils, monocytes and B cells (data not shown) and lead to cell activation assessed by neutrophil degranulation (MMP9 release) and monocyte TF expression (data not shown). To examine the contribution of complement activation in monocyte TF expression, WB was pre-incubated with α-C1q, α-C5 or IV.3 (a monoclonal antibody to FcγRIIA) or isotype controls prior to addition of HIT ULICs. As shown in Figure 2, the classical pathway inhibitor, α-C1q Ab markedly diminished TF expression (about 70% reduction; p<0.001 vPF4/H/ KKO), as did IV.3 (about 85% reduction; p<0.001 vPF4/H/ KKO) but not α-C5 Ab or ISO antibodies, demonstrating: 1) FcγRIIA independent mechanism of monocyte TF expression and 2) a requirement for proximal rather than terminal complement pathway components in the induction of monocyte TF. We next asked if complement activation facilitates binding of ULICs and promotes subsequent ULIC engagement of FcγRIIA. To examine complement dependent binding of HIT ULICs, we incubated WB with α-C1q Ab prior to addition of KKO ULICs and measured ULIC binding to monocytes and TF expression. As shown in Figure 3, classical pathway inhibition markedly reduced cell-surface IgG (Figure 3A) and monocyte TF expression (Figure 3B). The effects of complement inhibition could not be overcome with increasing amounts of KKO IgG (2-4 fold excess). We observed significant co-localization of complement with KKO ULICs in a cremaster-laser injury model in "HIT mice" and in in situ thrombi formed in uninjured vessels (data not shown). Together, these studies demonstrate an independent role for complement activation in regulating the binding and procoagulant effects of HIT ULICs and identify new non-anticoagulant therapeutic targets that could improve clinical outcomes in this otherwise potentially devastating thrombotic disorder. Disclosures Arepally: Novartis: Consultancy; Alexion: Other; Annexon Biosciences: Consultancy, Other; Veralox Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biokit: Consultancy, Patents & Royalties; Apotex: Consultancy, Research Funding.

Download Full-text

Complement activation during an active cytomegalovirus infection after renal transplantation: Due to circulating immune complexes or alternative pathway activation?

Clinical Immunology and Immunopathology ◽

10.1016/0090-1229(89)90226-2 ◽

1989 ◽

Vol 50 (1) ◽

pp. 109-121 ◽

Cited By ~ 13

Author(s):

Willem J. van Son ◽

Wim van der Bij ◽

Adam M. Tegzess ◽

Jetske Anema ◽

Marijke van der Giessen ◽

...

Keyword(s):

Renal Transplantation ◽

Complement Activation ◽

Immune Complexes ◽

Cytomegalovirus Infection ◽

Alternative Pathway ◽

Circulating Immune Complexes ◽

Pathway Activation

Download Full-text

Alternative Pathway Is Essential for Glomerular Complement Activation and Proteinuria in a Mouse Model of Membranous Nephropathy

Frontiers in Immunology ◽

10.3389/fimmu.2018.01433 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 19

Author(s):

Wentian Luo ◽

Florina Olaru ◽

Jeffrey H. Miner ◽

Laurence H. Beck ◽

Johan van der Vlag ◽

...

Keyword(s):

Mouse Model ◽

Complement Activation ◽

Membranous Nephropathy ◽

Alternative Pathway

Download Full-text

Minimal Role for the Alternative Pathway in Complement Activation By HIT Immune Complexes

Blood ◽

10.1182/blood-2021-150946 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2076-2076

Author(s):

Ayiesha Barnes ◽

Sanjay Khandelwal ◽

Simone Sartoretto ◽

Grace M Lee ◽

Sooho Myoung ◽

...

Keyword(s):

Cell Surface ◽

Complement Activation ◽

Immune Complexes ◽

Whole Blood ◽

Alternative Pathway ◽

Surface Deposition ◽

Cellular Activation ◽

C1 Esterase Inhibitor ◽

Complement Inhibitors ◽

Esterase Inhibitor

Abstract Background: Recent studies show that ultra-large immune complexes consisting of IgG and platelet factor 4 and heparin (P+H) potently activate complement and facilitate complement dependent activation of cellular FcgRIIA (PMID 34189574). In whole blood assays using KKO, a monoclonal anti-PF4/heparin antibody, or antibodies from patients with heparin induced thrombocytopenia (HIT), we showed that classical pathway (CP) inhibition reduced immune complex-mediated complement activation (C3c and soluble C5b-9 generation), cell surface deposition of immune complexes and cellular activation. Aims: As previous studies suggest that the alternative pathway (AP) provides significant amplification (>80%) of the CP pathway, (PMID: 15544620) we compared the effects of AP, CP, and CP/AP inhibitors by KKO and HIT immune complexes in whole blood. Methods: Inhibitors of the CP (BBK32, a borrelia protein inhibitor to C1r), AP (anti-factor B antibody (α-fB), or Factor D (fD inhibitor or fD-INH) Alexion Pharmaceuticals, Boston, MA) or combined AP/CP (C1-esterase inhibitor, C1-INH, Berinert, CSL Behring; or soluble complement receptor 1, sCR1, Alexion) were tested in hemolytic assays of CP or AP to confirm pathway specificity. To examine effects of CP or AP inhibition on complement activation by immune complexes consisting of KKO or HIT IgG, whole blood was pre-incubated with CP, AP or CP/AP inhibitors prior to addition of P+H ± KKO/HIT IgG or isotype controls. WB was incubated for 45 minutes at 37ºC followed by addition of 10mM EDTA to quench further complement activation. Complement activation products (C3c and sC5b-9) and neutrophil degranulation (MMP9) markers were measured using commercial immunoassays. Effects of complement inhibitors on cellular deposition of immune complexes was examined by flow, using previously described methods (PMID 34189574) using fluorescently labeled anti-C3c antibody (Quidel, San Diego, CA) and anti-mouse or human IgG (Biolegend, San Diego, CA). Results: Consistent with prior publications (PMID: 26808924), BBK32 showed marked reduction CP, but not AP-dependent hemolytic assays. The converse was true of AP inhibitors: α-fB and fD-INH prevented AP-dependent, but not CP-dependent hemolysis (data not shown). C1-INH and sCR1 showed activity in both CP- and AP-dependent assays. The CP or CP/AP inhibitors showed potent inhibition of C3c and sC5b-9 generation by KKO and HIT immune complexes, while AP inhibitors had no effect (Figure A for KKO C3c generation; and Table 1 for KKO/HIT C3c generation; sC5b-9 data not shown). For a given CP or CP/AP inhibitor, the concentrations leading to 50% inhibition (IC 50) were generally comparable for KKO and HIT immune complexes for C3c (Figure A and Table 1) and sC5b-9 generation (data not shown), with potency as follows: C1-INH>>BBK32>sCR1 (Table 1). On the other hand, the AP inhibitors, α-fB and fD-INH, showed no inhibitory activity in C3c (Figure A and Table 1)/sC5b-9 (data not shown) generation by KKO or HIT ULICs. As our recent studies indicate that complement activation is critical to cell surface deposition of immune complexes and cellular activation via FcgRIIA, we examined effects of complement inhibitors on IC deposition on B-cells and MMP9 release from neutrophils. CP or CP/AP inhibitors, but not AP inhibitors, reduced cell surface binding of immune complexes (Figure B) as well as MMP9 release (Figure C and Table 1). Conclusion: Together, these studies demonstrate that the AP has a minimal role in supporting complement activation by KKO/HIT ULICs. Future studies should examine CP inhibition as a therapeutic strategy for modulating the cellular activating effects of HIT antibodies. To what extent these findings apply to other immune complexes and/or CP activators requires further study. Funding Agency: NIH HL151730; α-fB antibody, fD inhibitor and sCR1 was provided by Alexion Pharmaceuticals, Boston, MA. BBK32 was provided by Dr. Brandon Garcia, East Carolina University, Greenville, NC. Figure 1 Figure 1. Disclosures Cines: Dova: Consultancy; Rigel: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board. OffLabel Disclosure: C1-esterase inhibitor off label for HIT

Download Full-text