Myeloperoxidase influences the complement regulatory activity of complement factor H

Myeloperoxidase influences the complement regulatory activity of complement factor H Abstract Objective The interaction between neutrophils and activation of alternative complement pathway plays a critical role in the pathogenesis of ANCA-associated vasculitis (AAV). MPO, which can be released from ANCA-stimulated neutrophils, was recently demonstrated to be capable of activating the alternative complement pathway. Here we aimed to investigate the interaction between MPO and factor H (FH), a key regulator of the alternative pathway, and its effect on the functional activities of FH. Methods Detection of FH and MPO on neutrophil extracellular traps (NETs) induced by serum from AAV patients and in kidney biopsies of AAV patients was performed by immunostaining. In vitro binding between MPO and FH was examined by ELISA and surface plasmon resonance. The influence of MPO on the complement regulatory activity of FH was further assessed. Results FH deposited and co-localized with MPO in NETs. In kidney biopsies from AAV patients, MPO was closely adjacent to FH in glomerular capillaries. We demonstrated that MPO binds to FH with an apparent nanomolar affinity and identified short consensus repeats 1–4 of FH as the major binding sites. In terms of functional analysis, MPO inhibited the interaction between FH and C3b and the decay-accelerating activity of FH. The fluid phase and surface cofactor activities of FH upon C3b inactivation were inhibited by MPO. Conclusion Our findings indicate that MPO binds to FH and influences the complement regulatory activity of FH. MPO-FH interaction may participate in the pathogenesis of AAV by contributing to activation of the alternative complement pathway. complement, factor H, MPO Rheumatology key messages MPO co-localized with factor H in neutrophil extracellular traps and was adjacent to it in glomeruli of ANCA patients. MPO binds to factor H, the N-terminal domains 1–4 serving as the major binding sites. MPO inhibits the complement regulatory activity of factor H. Introduction ANCA-associated vasculitis (AAV) comprises a group of systemic autoimmune diseases characterized by circulating autoantibodies against constituents of neutrophils, especially PR3 and MPO. Accumulating evidence from both animal studies and clinical studies has demonstrated the critical role of alternative complement pathway activation in the pathogenesis of AAV [1–8]. Activation of the alternative complement pathway amplifies the recruitment, priming and activation of neutrophils, creating a self-amplifying inflammatory loop that results in destructive necrotizing vascular injury [3, 9, 10]. Unlike the classic and lectin pathways, the alternative pathway of the complement system is characterized by spontaneous activation and self-amplification and is tightly regulated by several fluid-phase and cell-bound complement regulators under normal circumstances. How the alternative complement pathway is activated in AAV has not yet been fully elucidated. A series of in vitro studies showed that ANCA-stimulated neutrophils release substances that activate the complement system in serum [1, 3]. Interestingly, a recent study showed that MPO, the main ANCA autoantigen in neutrophils, is capable of activating the alternative complement pathway by interacting with properdin, the only positive regulator of the alternative pathway [11]. Meanwhile, our previous study found that MPO could block the binding between monomeric CRP (mCRP) and factor H, which might inhibit the negative regulation of alternative complement activation in AAV [12]. Complement factor H, composed of 20 short consensus repeats (SCRs), is a key regulator of the alternative pathway that strictly controls the alternative complement activation in circulation and at the host cell surface by accelerating the decay of C3 convertase (C3bBb) and by acting as a cofactor for factor I–mediated cleavage of C3b [13, 14]. The four N-terminal domains mediate complement regulation and the two C-terminal domains are relevant for host cell attachment [15–17]. Our recent studies suggested a potential role of complement factor H in the development of AAV [18, 19]. These led us to raise the question of whether MPO interacts with FH and influences the functional activity of FH. To address this issue, we characterized the staining of MPO and FH in neutrophil extracellular traps (NETs) and in kidney biopsies from patients with AAV. Then we explored the binding between MPO and FH and further investigated the influence of MPO on the complement regulatory activity of FH. Methods Immunofluorescence Increasing evidence has suggested a pathogenic role of NETs in the development of AAV. In particular, NETs induced by ANCA have been demonstrated to be capable of activating the alternative complement pathway [20]. Therefore deposition of FH on NETs was examined in the current study. NETs were induced using serum of patients with AAV, as previously described [21]. Briefly, neutrophils were isolated from heparinized whole blood of healthy blood donors. Neutrophils were seeded on eight-well chamber slides in Roswell Park Memorial Institute 1640 supplemented with 0.5% heat-inactivated foetal bovine serum at a density of 105 cells per well and allowed to adhere for 1 h at 37°C. Then, neutrophils were treated with serum of patients with AAV at a final concentration of 6% for 3 h at 37°C for NETs induction [21], followed by fixing with 4% paraformaldehyde. After washing with PBS, the samples were permeabilized for 1 min with 0.5% Triton X-100 in PBS. MPO and FH were stained with mouse anti-human MPO mAb (Abcam, Cambridge, MA, USA) and goat anti-human FH polyclonal antibody (Calbiochem, Darmstadt, Germany), respectively. Isotype controls for staining antibodies were performed by incubating with normal mouse IgG1 (Abcam) and normal goat IgG (Calbiochem). Fluorescent detection was achieved by incubation with Cy3-conjugated donkey anti-mouse IgG and Alexa Fluor 488–conjugated donkey anti-goat IgG. DNA was stained with 4′,6-diamidino-2-phenylindole (ZSGB-BIO, Beijing, China). Immunofluorescence staining was visualized in a confocal microscope (Carl Zeiss, Oberkochen, Germany). To examine the deposition of MPO and FH in the kidney, paraffin-fixed sections (2 μm) from renal biopsy specimens of patients with AAV were dewaxed, rehydrated and pre-treated with pepsin for antigen retrieval. Renal tissues obtained from the normal part of nephrectomized kidneys (because of renal carcinoma) were used as normal controls and tissue from a patient with diabetic nephropathy was included as the disease control. After blocking with PBS containing 3% BSA for 1 h, the sections were incubated with primary antibodies against human FH and MPO or an endothelial marker, CD34 (Abcam), overnight at 4°C followed by the corresponding secondary antibodies as described above. As isotype controls, primary antibodies were replaced by normal goat IgG with mouse IgG1 or normal rabbit IgG (Sigma, St Louis, MO. USA), respectively. Informed consent was obtained from each participant. The study was in compliance with the Declaration of Helsinki and was approved by the ethics committees of Peking University First Hospital. Interaction of MPO with FH and recombinant FH fragments measured by ELISA Microtitre plate wells were coated with 2 μg/ml MPO in PBS by overnight incubation at 4°C. A 0.2% gelatin was coated as negative controls. Non-specific binding to the plates was blocked with 0.2% gelatin. After washing, increasing concentrations of FH were added to the wells followed by detection using a polyclonal antibody against FH and then alkaline phosphatase–conjugated rabbit anti-goat IgG (Sigma). P-nitrophenyl phosphate (Sigma) substrate solution was used to visualize antibody binding and the absorbance was read at 405 nm. To identify the binding site for MPO on FH, full-length FH and recombinant FH fragments consisting of SCRs 1–4, 5–6, 7, 8–10, 11–14, 15–18 and 19–20 (GenScript, Piscataway, NJ, USA) were added to MPO-coated microtitre plates at the same molar concentrations (100 nM). Wells coated with 0.2% gelatin were set as negative controls. Bound FH and FH fragments were detected using goat anti-human FH antibody, which has been demonstrated to recognize the full-length FH and these SCRs of FH (see supplementary Fig. S1, available at Rheumatology online) [22]. Bound SCR19–20 was also detected using the mAb C18 (Enzo Life Sciences, Lörrach, Germany), a mAb against SCR20 of FH [23]. Surface plasmon resonance (SPR) analysis of MPO binding to FH The interaction between MPO and FH as well as FH fragments was further characterized by SPR in a Biacore T200 using S series CM5 sensor chips (GE Healthcare, Chicago, IL, USA) and analysed with the Biacore T200 evaluation software. MPO was immobilized on a CM5 chip using the amine-coupling procedure. FH and recombinant fragments of FH were allowed to flow across the chip in Tris buffer containing 10 mM Tris and 140 mM NaCl, pH 7.4 with 0.05% surfactant P-20. As controls, all binding tests were also performed using a blank chip activated and deactivated under identical conditions. After each binding experiment, the surface was regenerated by a short injection of 0.5 M NaCl. The binding kinetics of FH and the identified FH fragments for MPO binding were also analysed using chips immobilized with FH and relevant FH fragments, respectively. A chip coupled with an unrelated protein (gelatin) was used as the negative control. The surface was regenerated with 10 mM glycine-HCl, pH 2.0. Influence of MPO on the interaction between FH and C3b According to our previous findings, the mean concentration of MPO in the supernatants of ANCA activated neutrophils was ∼2 μg/ml [12]. Moreover, at this concentration, MPO largely inhibited the binding between mCRP and factor H [12]. Therefore a final concentration of 2 μg/mL MPO was applied in further experiments. Microtitre plate wells were coated with 4 μg/ml C3b (Calbiochem) in PBS overnight at 4°C. Serial dilutions of FH were incubated with 2 μg/ml MPO or buffer for 1 h at 37°C. In some experiments, 4 μg/ml FH were incubated with serial dilutions of MPO covering 2 μg/ml. After washing the plate, samples were transferred to individual wells for 1 h at 37°C. Bound FH was detected as described above. C3 convertase decay assay Solid-phase alternative pathway C3 convertase was assembled on immobilized C3b in microtitre plate wells, as described previously [24]. Decay acceleration was analysed by the addition of increasing concentrations (2.5, 5 and 10 μg/ml) of FH or human serum albumin as a control for 30 min at 37°C. To assess the influence of MPO on FH decay-accelerating activity, FH was pre-incubated with or without 2 μg/ml MPO for 1 h at 37°C. The remaining intact convertase was determined using a goat anti-factor B antiserum (Calbiochem). Cofactor assays To investigate whether MPO influences the cofactor activity of FH, 4 μg/ml FH with or without 2 μg/ml MPO were incubated with C3b (50 μg/ml) and factor I (10 μg/ml) for 10 min at 37°C in a final volume of 20 μl [25]. The reactions were stopped by adding reducing loading buffer with 5% β-mercaptoethanol. Samples were loaded onto 11% SDS-PAGE gels, separated by electrophoresis and subjected to western blot. C3 fragments were revealed using rabbit polyclonal antibody to human C3c (Dako, Glostrup, Denmark). Based on the previous study, the full-length FH is hypothesized to adopt, preferentially, a latent conformation accounting for low affinity for C3b, but a higher-affinity ‘activated’ conformation is stabilized by interaction with C3b and molecular markers on a self-surface [26]. Therefore the effect of MPO on the cofactor activity of FH on surfaces was analysed using the human basement membrane extract MaxGel (Sigma) and human renal glomerular endothelial cells (HRGECs; ScienCell Research Laboratories, San Diego, CA, USA), respectively. To assay cofactor activity on MaxGel [27], 40 μl of 1 μM FH pre-incubated with 2 μg/ml MPO or buffer were added to wells coated with MaxGel [diluted 1:40 in Tris-buffered saline (TBS) containing 2 mM CaCl2 and 1 mM MgCl2] for 1 h at 37°C. After washing, 50 μg/ml C3b and 10 μg/ml factor I diluted in TBS were added in 40 μl to the wells and incubated for 30 min at 37°C. C3 cleavage products in the supernatants were identified, as described above. In order to investigate the effect on the host cell surface, the cofactor activity of FH on HRGECs was measured according to the previously described method [27], with some minor modifications. Briefly, endothelial cells growing on 96-well cell culture plates were incubated with 1 μM FH pre-incubated with 2 μg/ml MPO or buffer in a final volume of 40 μl in Dulbecco’s PBS at 37°C for 1 h. After washing, cells were incubated with 50 μg/ml C3b and 10 μg/ml factor I in 40 μl for 30 min at 37°C. Supernatants were analysed for C3b cleavage, as described above. Haemolysis assays To assess the influence of MPO on the protective ability of FH against complement-mediated haemolysis of sheep erythrocytes, FH pre-incubated with MPO or buffer was added to sheep erythrocytes in the presence of 4 μg mAb OX24 (binding to SCR5 of FH) with 15% normal human serum in a final volume of 200 μl, as described previously [23]. The reactions were incubated at 37°C for 30 min, the erythrocytes were sedimented by centrifugation and the released haemoglobin was measured in the supernatants at 414 nm. C3a and C5a in the supernatants were measured using commercial ELISA kits according to the manufacturer’s instructions (Quidel, San Diego, CA, USA). Statistical analysis Data were presented as mean (s.d.). Statistical differences between groups were determined by t test or analysis of variance as appropriate. P-values <0.05 were considered statistically significant. Analysis was performed with SPSS statistical software (version 13.0; SPSS, Chicago, IL, USA). Results Immunostaining of FH and MPO in NETs and renal biopsies of AAV NETs were induced by serum from patients with AAV, as determined by decondensed extracellular DNA decorated with extracellular MPO. We found that FH deposited in NETs and merged with the staining of MPO (Fig. 1). The renal biopsies of patients with AAV demonstrated positive staining of FH distributed along endothelium cells of the glomeruli, co-localizing with the staining of endothelial cells (CD34). MPO appeared to decorate the glomerular capillaries and was closely adjacent to the staining of FH. In some areas of the glomerulus, co-localization of MPO and FH on endothelial cells was observed (Fig. 2). Nevertheless, glomerulus from normal controls and glomerulus from a patient with diabetic nephropathy demonstrated little staining of FH and no intraglomerular MPO with intact CD34+ endothelial cells. Staining with isotype controls is shown in supplementary Fig. S2, available at Rheumatology online. Fig. 1 View largeDownload slide Immunostaining of FH and MPO in NETs NETs were induced by incubating neutrophils isolated from healthy donors with serum from AAV patients. (A–C) NETs released from ANCA-activated neutrophils demonstrated positive staining of FH, co-localizing with the staining of MPO. (D and E) Isotype controls for staining antibodies were performed by incubating with normal goat IgG and mouse IgG1. Representative images from three independent experiments are shown. DAPI: 4′,6-diamidino-2-phenylindole. Fig. 1 View largeDownload slide Immunostaining of FH and MPO in NETs NETs were induced by incubating neutrophils isolated from healthy donors with serum from AAV patients. (A–C) NETs released from ANCA-activated neutrophils demonstrated positive staining of FH, co-localizing with the staining of MPO. (D and E) Isotype controls for staining antibodies were performed by incubating with normal goat IgG and mouse IgG1. Representative images from three independent experiments are shown. DAPI: 4′,6-diamidino-2-phenylindole. Fig. 2 View largeDownload slide Immunostaining of FH and MPO in renal specimens (A) Glomerulus from patients with AAV demonstrated positive staining of FH and MPO. MPO was closely adjacent to FH and there were areas showing co-localization of MPO and FH (arrow). (B and C) Glomerulus from normal controls and glomerulus from a patient with diabetic nephropathy demonstrated scanty staining of FH and no intraglomerular MPO. (D) FH was distributed along the capillary loops of endothelial cells in glomeruli of patients with AAV, co-localized with the staining of endothelial cells (CD34). (E and F) Glomerulus from normal controls and glomerulus from diabetic nephropathy showed scarce deposition of FH with intact CD34+ endothelial cells. Representative images from three independent experiments are shown. Fig. 2 View largeDownload slide Immunostaining of FH and MPO in renal specimens (A) Glomerulus from patients with AAV demonstrated positive staining of FH and MPO. MPO was closely adjacent to FH and there were areas showing co-localization of MPO and FH (arrow). (B and C) Glomerulus from normal controls and glomerulus from a patient with diabetic nephropathy demonstrated scanty staining of FH and no intraglomerular MPO. (D) FH was distributed along the capillary loops of endothelial cells in glomeruli of patients with AAV, co-localized with the staining of endothelial cells (CD34). (E and F) Glomerulus from normal controls and glomerulus from diabetic nephropathy showed scarce deposition of FH with intact CD34+ endothelial cells. Representative images from three independent experiments are shown. Binding between FH and MPO The binding between FH and MPO was determined by ELISA using a polyclonal antibody against FH. An increasing concentration of FH showed dose-dependent binding to coated MPO (Fig. 3A). To identify the binding sites for MPO on FH, we assessed the binding of recombinantly expressed FH fragments that span through the whole molecule to coated MPO. SCR1–4 of FH was identified as containing the major binding site for MPO (Fig. 3B). To rule out the possibility that FH was interacting with traces of DNA that still could not be completely isolated from MPO during purification, MPO was treated with 0–10 units of DNase-1 (Sigma) prior to FH incubation; no obvious difference in FH binding was detected (see supplementary Fig. S3, available at Rheumatology online). Considering that MPO serves as an autoantigen for ANCA in patients with MPO-ANCA-positive vasculitis, we assessed whether its interaction with FH is inhibited by ANCA. As expected, this interaction was not diminished in the presence of ANCA (see supplementary Fig. S4, available at Rheumatology online). Fig. 3 View largeDownload slide Analysis of MPO–FH interaction by microtitre plate binding assays and SPR (A) Dose-dependent binding of FH to MPO assayed by ELISA. A representative experiment of three performed is shown. (B) Mapping of the MPO–FH interaction sites by ELISA. Full-length FH and FH fragments were incubated with MPO-coated wells. The values represent mean (s.d.) derived from three independent experiments. (C) The SCR1–4 of FH was identified as the major binding site for MPO by SPR. Full-length FH and FH fragments were allowed to flow across an MPO immobilized chip; the signal obtained on a blank chip was subtracted. (D and E) SPR analysis of the binding kinetics of FH and SCR1–4 to the MPO immobilized chip. The affinity constant was 5.51 × 10−9 M and 5.95 × 10−9 M, respectively. (F and G) MPO bound to FH and SCR1–4 coupled chips with similar affinities, which was 1.28 × 10−9 M and 3.66 × 10−10 M, respectively. All SPR experiments were repeated twice independently. Fig. 3 View largeDownload slide Analysis of MPO–FH interaction by microtitre plate binding assays and SPR (A) Dose-dependent binding of FH to MPO assayed by ELISA. A representative experiment of three performed is shown. (B) Mapping of the MPO–FH interaction sites by ELISA. Full-length FH and FH fragments were incubated with MPO-coated wells. The values represent mean (s.d.) derived from three independent experiments. (C) The SCR1–4 of FH was identified as the major binding site for MPO by SPR. Full-length FH and FH fragments were allowed to flow across an MPO immobilized chip; the signal obtained on a blank chip was subtracted. (D and E) SPR analysis of the binding kinetics of FH and SCR1–4 to the MPO immobilized chip. The affinity constant was 5.51 × 10−9 M and 5.95 × 10−9 M, respectively. (F and G) MPO bound to FH and SCR1–4 coupled chips with similar affinities, which was 1.28 × 10−9 M and 3.66 × 10−10 M, respectively. All SPR experiments were repeated twice independently. To further confirm and characterize the interaction between MPO and FH, SPR was performed. The injection of FH on an MPO immobilized chip showed dose-dependent binding with an apparent KD of 5.51 × 10−9 M. At physiological pH and ionic strength, FH-MPO association and dissociation constants were 4.49 × 104/Ms (s.d. 0.03 × 104) and 2.47 × 10−4/s (s.d. 0.05 × 10−4), respectively. Using the same chip, SCR1–4 of FH was confirmed to contain the major binding site for MPO with an apparent KD of 5.95 × 10−9 M, which was comparable to the full-length FH (Fig. 3C–E). The calculated apparent association and dissociation constants were 9.68 × 104/Ms (s.d. 0.09 × 104) and 5.76 × 10−4/s (s.d. 0.03 × 10−4 ), respectively. Consistently, MPO bound to FH and SCR1–4 coupled chips with similar affinities, which was 1.28 × 10−9 M and 3.66 × 10−10 M, respectively (Fig. 3F and G). MPO inhibits the interaction of FH with C3b and the functional activity of FH in the regulation of C3b Interaction of FH with C3b is essential for the regulatory activities of FH, as it mediates the decay-accelerating and cofactor activities of FH. Therefore we first investigated whether the FH–C3b interaction could be altered in the presence of MPO. MPO inhibited the binding of FH to C3b in a dose-dependent manner. By incubating increasing concentrations of FH mixed with 2 μg/ml MPO in C3b-coated wells, we found the FH bound dose-dependently to C3b, but the interaction between FH and C3b was largely inhibited in the presence of MPO (P < 0.001; Fig. 4A and B). Then we tested the influence of the MPO–FH interaction on the decay-accelerating activity of FH. The results showed that FH accelerated the decay of C3 convertase (C3bBb), which was assembled on immobilized C3b, in a dose-dependent manner (Fig. 4C). In the presence 2 μg/ml MPO, the decay-accelerating activity of FH was significantly weakened [0.56 (s.d. 0.02) vs 0.70 (s.d. 0.04), P = 0.004 for FH at 10 μg/ml; 0.88 (s.d. 0.01) vs 1.02 (s.d. 0.04), P = 0.004 for FH at 5 μg/ml] (Fig. 4D). To explore whether MPO interferes with the cofactor activity of CFH upon C3b inactivation, the fluid phase cofactor activity of FH in the presence of MPO was assayed. MPO inhibited the cofactor activity of FH, as evidenced by the increased density of the uncleaved α′ 108 band and the decreased density of the cleaved α′ 43 band (Fig. 5A). Densitometric analyses of the relative intensity of the α′ 43 band showed that the fluid phase cofactor activity of FH was decreased by ∼40% in the presence of 2 μg/ml MPO [100% (s.d. 0.00) vs 58.98% (s.d. 5.13), P < 0.001] (Fig. 5B). We further found that cofactor activity of FH on human basement membrane extract MaxGel and HRGECs were also inhibited in the presence of 2 μg/ml MPO. Densitometric analyses showed that the cofactor activity of FH on such surfaces was decreased by ∼30% in the presence of 2 μg/ml MPO [100% (s.d. 0.00) vs 69.38% (s.d. 3.35), P < 0.001; 100% (s.d. 0.00) vs 70.79% (s.d. 2.28), P < 0.001, respectively] (Fig. 5C–F). Fig. 4 View largeDownload slide Influence of MPO on FH–C3b interaction and the decay-accelerating activity of FH (A) MPO inhibited the binding of FH to C3b in a dose-dependent manner (***P < 0.001, Student’s t test). The values represent mean (s.d.) derived from three independent experiments. (B) Binding of FH to coated C3b was inhibited in the presence of 2 μg/ml MPO (P < 0.001, two-way analysis of variance). Shown is the mean (s.d.) of three independent experiments. (C) Dose-dependent convertase decay-accelerating effects of FH; human serum albumin (HSA) was set as a control. (D) In the presence 2 μg/ml MPO, the decay-accelerating activity of FH was significantly weakened. The bars and error bars represent the mean (s.d.) of the remaining C3 convertase after decay by FH. Results are derived from three independent experiments. Fig. 4 View largeDownload slide Influence of MPO on FH–C3b interaction and the decay-accelerating activity of FH (A) MPO inhibited the binding of FH to C3b in a dose-dependent manner (***P < 0.001, Student’s t test). The values represent mean (s.d.) derived from three independent experiments. (B) Binding of FH to coated C3b was inhibited in the presence of 2 μg/ml MPO (P < 0.001, two-way analysis of variance). Shown is the mean (s.d.) of three independent experiments. (C) Dose-dependent convertase decay-accelerating effects of FH; human serum albumin (HSA) was set as a control. (D) In the presence 2 μg/ml MPO, the decay-accelerating activity of FH was significantly weakened. The bars and error bars represent the mean (s.d.) of the remaining C3 convertase after decay by FH. Results are derived from three independent experiments. Fig. 5 View largeDownload slide MPO inhibits the cofactor activity of FH (A) The fluid phase cofactor activity of FH for C3b cleavage was inhibited in the presence of MPO, as evidenced by the increased density of the uncleaved α′ 108 band and the decreased density of the cleaved α′ 43 band. A representative blot of three experiments is shown. (B) Densitometric analysis of the relative intensity of the cleavage fragment of C3b and the ratio of the α′ 43 band to the α′ 108 band. Data are presented as the mean (s.d.) of the percentage of C3b generation achieved with commercial FH (100%). The results of three independent experiments are shown. (C) The cofactor activity of FH on MaxGel was inhibited by MPO. (D) C3b cleavage was densitometrically quantified as described above. Results are derived from three independent experiments. (E) The cofactor activity of FH on HRGECs was inhibited by MPO. (F) Densitometric analysis of C3b proteolysis as described. Shown is the mean (s.d.) of three independent experiments (***P < 0.001). Fig. 5 View largeDownload slide MPO inhibits the cofactor activity of FH (A) The fluid phase cofactor activity of FH for C3b cleavage was inhibited in the presence of MPO, as evidenced by the increased density of the uncleaved α′ 108 band and the decreased density of the cleaved α′ 43 band. A representative blot of three experiments is shown. (B) Densitometric analysis of the relative intensity of the cleavage fragment of C3b and the ratio of the α′ 43 band to the α′ 108 band. Data are presented as the mean (s.d.) of the percentage of C3b generation achieved with commercial FH (100%). The results of three independent experiments are shown. (C) The cofactor activity of FH on MaxGel was inhibited by MPO. (D) C3b cleavage was densitometrically quantified as described above. Results are derived from three independent experiments. (E) The cofactor activity of FH on HRGECs was inhibited by MPO. (F) Densitometric analysis of C3b proteolysis as described. Shown is the mean (s.d.) of three independent experiments (***P < 0.001). Influence of MPO on the protective ability of FH against complement-mediated attack on sheep erythrocytes We further investigated whether MPO interferes with the protective ability of FH against complement-mediated haemolysis of sheep erythrocytes. Sheep erythrocytes lysis was induced in normal human plasma by mAb OX24, which inhibits the N-terminal complement regulatory domains. A dose-dependent inhibition of this lysis was achieved when FH was added (Fig. 6A). The addition of MPO did not interfere with the protective ability of FH against sheep erythrocytes lysis induced by mAb OX24 (Fig. 6B). However, levels of C3a and C5a in the supernatant were significantly higher when FH was pre-incubated with MPO (Fig. 6C and D). Fig. 6 View largeDownload slide Influence of MPO on the protective ability of FH against complement-mediated attack on sheep erythrocytes (A) Sheep erythrocytes lysis was induced by the FH mAb OX24 with 15% normal human serum. FH protects sheep erythrocytes from haemolysis induced by mAb OX24 in a dose-dependent manner. (B) MPO did not interfere with the protective ability of FH against sheep erythrocytes lysis induced by mAb OX24. (C and D) Pre-incubation of FH with MPO resulted in more C3a and C5a production in the supernatants. Data are mean (s.d.) from three independent experiments. Fig. 6 View largeDownload slide Influence of MPO on the protective ability of FH against complement-mediated attack on sheep erythrocytes (A) Sheep erythrocytes lysis was induced by the FH mAb OX24 with 15% normal human serum. FH protects sheep erythrocytes from haemolysis induced by mAb OX24 in a dose-dependent manner. (B) MPO did not interfere with the protective ability of FH against sheep erythrocytes lysis induced by mAb OX24. (C and D) Pre-incubation of FH with MPO resulted in more C3a and C5a production in the supernatants. Data are mean (s.d.) from three independent experiments. Discussion The interaction between neutrophils and activation of alternative complement pathway plays a pivotal role in the pathogenesis of AAV. An in vitro study showed that neutrophils activated by ANCA release factors capable of activating the complement system and generating C5a [3]. C5a further primes neutrophils for activation by ANCA [3, 9, 10], thus causing a self-amplification loop for ANCA-induced neutrophil activation. Among various neutrophil constituents that could be released upon activation, MPO was previously shown to be capable of activating the alternative complement pathway by interacting with properdin [11]. Our current study found that MPO interacts with the N-terminus of FH and inhibits the complement regulatory activity of FH, indicating a novel path for MPO in activating the complement system. Another interesting finding is that FH deposited in NETs and co-localized with MPO. By demonstrating that the complement regulatory activity of FH was inhibited by MPO, the current study further extended our previous finding that NETs released from ANCA-activated neutrophils are capable of activating the alternative complement pathway [20]. Moreover, by attracting FH binding, it might contribute to the decreased concentration of FH in the plasma of patients with AAV [18]. Based on our previous findings, decreased plasma levels of FH and deficient functional activities of FH were associated with disease activity and circulating levels of complement activation products in AAV [18, 19], indicating an important role of FH in disease development. Therefore our current finding indicates that the MPO–FH interaction may contribute to activation of the alternative complement pathway in AAV patients. Although, ANCA did not influence the binding of MPO to FH, we speculate that it would amplify the effects resulting from the MPO–FH interaction. By inhibiting FH, MPO contributes to the generation of more C5a, which would further prime neutrophils for activation by ANCA, resulting in more degranulation of MPO and thus causing a self-fuelling inflammatory amplification loop. In the renal specimens of patients with ANCA-associated glomerulonephritis, MPO was closely adjacent to FH localized to the endothelial surface of the capillary wall, which is consistent with the previous finding that MPO adheres to glomerular endothelial cells and is associated with the extent of endothelial injury in patients with ANCA-associated glomerulonephritis [28]. Although MPO is subendothelially concentrated [28, 29], there is considerable evidence demonstrating that MPO binds to endothelial cells and lasts for hours [29–31]. However, MPO would be transferred from the cell surface to cytoplasm, and possibly nuclear, through an internalization process by endothelial cells [30, 31]. It may account for the current observation that some areas of renal specimens from AAV patients showed co-localization of MPO and FH on endothelial cells, but in most areas of the glomeruli, MPO was subendothelially localized and closely adjacent to FH. Nevertheless, it could not exclude the possibility that the interaction of MPO with FH occurred on the glomerular endothelium. Moreover, given that MPO was able to mediate nitration of tyrosine residues during inflammatory processes [32, 33], and interestingly, nitration of tyrosines in factor H was recently reported to result in the loss of functional activities in interacting with C3b and cofactor activity for factor I–mediated cleavage of C3b [34], we speculate that MPO adhering to the glomerular endothelium might inhibit the negative regulation of alternative complement activation on endothelial cells. It extends the current understanding that MPO can mediate injury of glomerular capillary walls in AAV by direct oxidative damage and by triggering humoral and cellular immune response [35]. Although a significant inhibitory effect of MPO on FH-mediated inactivation of C3b on host cell surfaces was observed, no obvious effect can be detected in the haemolytic assays. However, we demonstrated that pre-incubation of FH with MPO resulted in more C3a and C5a production in the supernatants of haemolytic assays. Regarding the end product of complement activation, it is C5a, not C5b-9, that plays a critical role in the pathogenesis of AAV, as evidenced by the animal study [1, 8]. C5a not only primes neutrophils for activation by ANCA, but also amplifies the neutrophil–endothelial cell interactions on the surface of capillary walls [36]. Therefore we suggest that, by contributing to the generation of more C5a, MPO–FH interaction may contribute to the disease development of AAV. On the other hand, given that ceruloplasmin, the physiological inhibitor of MPO, exists in the serum, it is reasonable to speculate that it may present as a confounder for the result of the haemolysis assays. However, in patients with AAV, the inhibition of MPO by ceruloplasmin can be reversed by ANCA [37]. It would be of great interest to carry out in vivo studies to investigate the role of MPO on alternative complement activation and regulation in AAV. Overall, the effect of MPO’s interaction on FH cofactor activity and decay accelerating activity or C3a/C5a production is rather modest, which is a limitation of the current study. However, given the fact that the alternative complement pathway is characterized by self-amplification, it was suggested that the very nature of the complement system will amplify these small effects [25]. Considering that, along with AAV, alternative complement activation has been implicated in various neutrophil-mediated diseases, such as bacterial sepsis [38, 39], the interaction between MPO and FH and its effect on the complement regulatory activity of FH may not be limited in AAV. However, the biological significance of FH in bacterial sepsis is still controversial given that FH was previously reported to contribute to immune invasion of at least 10 pathogenic microbes [40–43]. Therefore it would be of great interest to investigate the role of the MPO–FH interaction in other diseases. In conclusion, MPO, which can be released from ANCA-stimulated neutrophils, binds to FH and inhibits the complement regulatory activity of FH. The MPO–FH interaction may participate in the pathogenesis of AAV by contributing to activation of the alternative complement pathway. Acknowledgements We are very grateful to Prof Ge Fu and Jing Wang from the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, for technical assistance on the surface plasmon resonance analysis. Funding: This work was supported by a grant from the National Key Research and Development Program (2016YFC0906102), three grants from the National Natural Science Fund (81400724, 81425008 and 81621092) and by a grant from the University of Michigan Health System and Peking University Health Sciences Center Joint Institute for Translational and Clinical Research. Disclosure statement: The authors have declared no conflicts of interest. 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Myeloperoxidase influences the complement regulatory activity of complement factor H

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Abstract

Abstract Objective The interaction between neutrophils and activation of alternative complement pathway plays a critical role in the pathogenesis of ANCA-associated vasculitis (AAV). MPO, which can be released from ANCA-stimulated neutrophils, was recently demonstrated to be capable of activating the alternative complement pathway. Here we aimed to investigate the interaction between MPO and factor H (FH), a key regulator of the alternative pathway, and its effect on the functional activities of FH. Methods Detection of FH and MPO on neutrophil extracellular traps (NETs) induced by serum from AAV patients and in kidney biopsies of AAV patients was performed by immunostaining. In vitro binding between MPO and FH was examined by ELISA and surface plasmon resonance. The influence of MPO on the complement regulatory activity of FH was further assessed. Results FH deposited and co-localized with MPO in NETs. In kidney biopsies from AAV patients, MPO was closely adjacent to FH in glomerular capillaries. We demonstrated that MPO binds to FH with an apparent nanomolar affinity and identified short consensus repeats 1–4 of FH as the major binding sites. In terms of functional analysis, MPO inhibited the interaction between FH and C3b and the decay-accelerating activity of FH. The fluid phase and surface cofactor activities of FH upon C3b inactivation were inhibited by MPO. Conclusion Our findings indicate that MPO binds to FH and influences the complement regulatory activity of FH. MPO-FH interaction may participate in the pathogenesis of AAV by contributing to activation of the alternative complement pathway. complement, factor H, MPO Rheumatology key messages MPO co-localized with factor H in neutrophil extracellular traps and was adjacent to it in glomeruli of ANCA patients. MPO binds to factor H, the N-terminal domains 1–4 serving as the major binding sites. MPO inhibits the complement regulatory activity of factor H. Introduction ANCA-associated vasculitis (AAV) comprises a group of systemic autoimmune diseases characterized by circulating autoantibodies against constituents of neutrophils, especially PR3 and MPO. Accumulating evidence from both animal studies and clinical studies has demonstrated the critical role of alternative complement pathway activation in the pathogenesis of AAV [1–8]. Activation of the alternative complement pathway amplifies the recruitment, priming and activation of neutrophils, creating a self-amplifying inflammatory loop that results in destructive necrotizing vascular injury [3, 9, 10]. Unlike the classic and lectin pathways, the alternative pathway of the complement system is characterized by spontaneous activation and self-amplification and is tightly regulated by several fluid-phase and cell-bound complement regulators under normal circumstances. How the alternative complement pathway is activated in AAV has not yet been fully elucidated. A series of in vitro studies showed that ANCA-stimulated neutrophils release substances that activate the complement system in serum [1, 3]. Interestingly, a recent study showed that MPO, the main ANCA autoantigen in neutrophils, is capable of activating the alternative complement pathway by interacting with properdin, the only positive regulator of the alternative pathway [11]. Meanwhile, our previous study found that MPO could block the binding between monomeric CRP (mCRP) and factor H, which might inhibit the negative regulation of alternative complement activation in AAV [12]. Complement factor H, composed of 20 short consensus repeats (SCRs), is a key regulator of the alternative pathway that strictly controls the alternative complement activation in circulation and at the host cell surface by accelerating the decay of C3 convertase (C3bBb) and by acting as a cofactor for factor I–mediated cleavage of C3b [13, 14]. The four N-terminal domains mediate complement regulation and the two C-terminal domains are relevant for host cell attachment [15–17]. Our recent studies suggested a potential role of complement factor H in the development of AAV [18, 19]. These led us to raise the question of whether MPO interacts with FH and influences the functional activity of FH. To address this issue, we characterized the staining of MPO and FH in neutrophil extracellular traps (NETs) and in kidney biopsies from patients with AAV. Then we explored the binding between MPO and FH and further investigated the influence of MPO on the complement regulatory activity of FH. Methods Immunofluorescence Increasing evidence has suggested a pathogenic role of NETs in the development of AAV. In particular, NETs induced by ANCA have been demonstrated to be capable of activating the alternative complement pathway [20]. Therefore deposition of FH on NETs was examined in the current study. NETs were induced using serum of patients with AAV, as previously described [21]. Briefly, neutrophils were isolated from heparinized whole blood of healthy blood donors. Neutrophils were seeded on eight-well chamber slides in Roswell Park Memorial Institute 1640 supplemented with 0.5% heat-inactivated foetal bovine serum at a density of 105 cells per well and allowed to adhere for 1 h at 37°C. Then, neutrophils were treated with serum of patients with AAV at a final concentration of 6% for 3 h at 37°C for NETs induction [21], followed by fixing with 4% paraformaldehyde. After washing with PBS, the samples were permeabilized for 1 min with 0.5% Triton X-100 in PBS. MPO and FH were stained with mouse anti-human MPO mAb (Abcam, Cambridge, MA, USA) and goat anti-human FH polyclonal antibody (Calbiochem, Darmstadt, Germany), respectively. Isotype controls for staining antibodies were performed by incubating with normal mouse IgG1 (Abcam) and normal goat IgG (Calbiochem). Fluorescent detection was achieved by incubation with Cy3-conjugated donkey anti-mouse IgG and Alexa Fluor 488–conjugated donkey anti-goat IgG. DNA was stained with 4′,6-diamidino-2-phenylindole (ZSGB-BIO, Beijing, China). Immunofluorescence staining was visualized in a confocal microscope (Carl Zeiss, Oberkochen, Germany). To examine the deposition of MPO and FH in the kidney, paraffin-fixed sections (2 μm) from renal biopsy specimens of patients with AAV were dewaxed, rehydrated and pre-treated with pepsin for antigen retrieval. Renal tissues obtained from the normal part of nephrectomized kidneys (because of renal carcinoma) were used as normal controls and tissue from a patient with diabetic nephropathy was included as the disease control. After blocking with PBS containing 3% BSA for 1 h, the sections were incubated with primary antibodies against human FH and MPO or an endothelial marker, CD34 (Abcam), overnight at 4°C followed by the corresponding secondary antibodies as described above. As isotype controls, primary antibodies were replaced by normal goat IgG with mouse IgG1 or normal rabbit IgG (Sigma, St Louis, MO. USA), respectively. Informed consent was obtained from each participant. The study was in compliance with the Declaration of Helsinki and was approved by the ethics committees of Peking University First Hospital. Interaction of MPO with FH and recombinant FH fragments measured by ELISA Microtitre plate wells were coated with 2 μg/ml MPO in PBS by overnight incubation at 4°C. A 0.2% gelatin was coated as negative controls. Non-specific binding to the plates was blocked with 0.2% gelatin. After washing, increasing concentrations of FH were added to the wells followed by detection using a polyclonal antibody against FH and then alkaline phosphatase–conjugated rabbit anti-goat IgG (Sigma). P-nitrophenyl phosphate (Sigma) substrate solution was used to visualize antibody binding and the absorbance was read at 405 nm. To identify the binding site for MPO on FH, full-length FH and recombinant FH fragments consisting of SCRs 1–4, 5–6, 7, 8–10, 11–14, 15–18 and 19–20 (GenScript, Piscataway, NJ, USA) were added to MPO-coated microtitre plates at the same molar concentrations (100 nM). Wells coated with 0.2% gelatin were set as negative controls. Bound FH and FH fragments were detected using goat anti-human FH antibody, which has been demonstrated to recognize the full-length FH and these SCRs of FH (see supplementary Fig. S1, available at Rheumatology online) [22]. Bound SCR19–20 was also detected using the mAb C18 (Enzo Life Sciences, Lörrach, Germany), a mAb against SCR20 of FH [23]. Surface plasmon resonance (SPR) analysis of MPO binding to FH The interaction between MPO and FH as well as FH fragments was further characterized by SPR in a Biacore T200 using S series CM5 sensor chips (GE Healthcare, Chicago, IL, USA) and analysed with the Biacore T200 evaluation software. MPO was immobilized on a CM5 chip using the amine-coupling procedure. FH and recombinant fragments of FH were allowed to flow across the chip in Tris buffer containing 10 mM Tris and 140 mM NaCl, pH 7.4 with 0.05% surfactant P-20. As controls, all binding tests were also performed using a blank chip activated and deactivated under identical conditions. After each binding experiment, the surface was regenerated by a short injection of 0.5 M NaCl. The binding kinetics of FH and the identified FH fragments for MPO binding were also analysed using chips immobilized with FH and relevant FH fragments, respectively. A chip coupled with an unrelated protein (gelatin) was used as the negative control. The surface was regenerated with 10 mM glycine-HCl, pH 2.0. Influence of MPO on the interaction between FH and C3b According to our previous findings, the mean concentration of MPO in the supernatants of ANCA activated neutrophils was ∼2 μg/ml [12]. Moreover, at this concentration, MPO largely inhibited the binding between mCRP and factor H [12]. Therefore a final concentration of 2 μg/mL MPO was applied in further experiments. Microtitre plate wells were coated with 4 μg/ml C3b (Calbiochem) in PBS overnight at 4°C. Serial dilutions of FH were incubated with 2 μg/ml MPO or buffer for 1 h at 37°C. In some experiments, 4 μg/ml FH were incubated with serial dilutions of MPO covering 2 μg/ml. After washing the plate, samples were transferred to individual wells for 1 h at 37°C. Bound FH was detected as described above. C3 convertase decay assay Solid-phase alternative pathway C3 convertase was assembled on immobilized C3b in microtitre plate wells, as described previously [24]. Decay acceleration was analysed by the addition of increasing concentrations (2.5, 5 and 10 μg/ml) of FH or human serum albumin as a control for 30 min at 37°C. To assess the influence of MPO on FH decay-accelerating activity, FH was pre-incubated with or without 2 μg/ml MPO for 1 h at 37°C. The remaining intact convertase was determined using a goat anti-factor B antiserum (Calbiochem). Cofactor assays To investigate whether MPO influences the cofactor activity of FH, 4 μg/ml FH with or without 2 μg/ml MPO were incubated with C3b (50 μg/ml) and factor I (10 μg/ml) for 10 min at 37°C in a final volume of 20 μl [25]. The reactions were stopped by adding reducing loading buffer with 5% β-mercaptoethanol. Samples were loaded onto 11% SDS-PAGE gels, separated by electrophoresis and subjected to western blot. C3 fragments were revealed using rabbit polyclonal antibody to human C3c (Dako, Glostrup, Denmark). Based on the previous study, the full-length FH is hypothesized to adopt, preferentially, a latent conformation accounting for low affinity for C3b, but a higher-affinity ‘activated’ conformation is stabilized by interaction with C3b and molecular markers on a self-surface [26]. Therefore the effect of MPO on the cofactor activity of FH on surfaces was analysed using the human basement membrane extract MaxGel (Sigma) and human renal glomerular endothelial cells (HRGECs; ScienCell Research Laboratories, San Diego, CA, USA), respectively. To assay cofactor activity on MaxGel [27], 40 μl of 1 μM FH pre-incubated with 2 μg/ml MPO or buffer were added to wells coated with MaxGel [diluted 1:40 in Tris-buffered saline (TBS) containing 2 mM CaCl2 and 1 mM MgCl2] for 1 h at 37°C. After washing, 50 μg/ml C3b and 10 μg/ml factor I diluted in TBS were added in 40 μl to the wells and incubated for 30 min at 37°C. C3 cleavage products in the supernatants were identified, as described above. In order to investigate the effect on the host cell surface, the cofactor activity of FH on HRGECs was measured according to the previously described method [27], with some minor modifications. Briefly, endothelial cells growing on 96-well cell culture plates were incubated with 1 μM FH pre-incubated with 2 μg/ml MPO or buffer in a final volume of 40 μl in Dulbecco’s PBS at 37°C for 1 h. After washing, cells were incubated with 50 μg/ml C3b and 10 μg/ml factor I in 40 μl for 30 min at 37°C. Supernatants were analysed for C3b cleavage, as described above. Haemolysis assays To assess the influence of MPO on the protective ability of FH against complement-mediated haemolysis of sheep erythrocytes, FH pre-incubated with MPO or buffer was added to sheep erythrocytes in the presence of 4 μg mAb OX24 (binding to SCR5 of FH) with 15% normal human serum in a final volume of 200 μl, as described previously [23]. The reactions were incubated at 37°C for 30 min, the erythrocytes were sedimented by centrifugation and the released haemoglobin was measured in the supernatants at 414 nm. C3a and C5a in the supernatants were measured using commercial ELISA kits according to the manufacturer’s instructions (Quidel, San Diego, CA, USA). Statistical analysis Data were presented as mean (s.d.). Statistical differences between groups were determined by t test or analysis of variance as appropriate. P-values <0.05 were considered statistically significant. Analysis was performed with SPSS statistical software (version 13.0; SPSS, Chicago, IL, USA). Results Immunostaining of FH and MPO in NETs and renal biopsies of AAV NETs were induced by serum from patients with AAV, as determined by decondensed extracellular DNA decorated with extracellular MPO. We found that FH deposited in NETs and merged with the staining of MPO (Fig. 1). The renal biopsies of patients with AAV demonstrated positive staining of FH distributed along endothelium cells of the glomeruli, co-localizing with the staining of endothelial cells (CD34). MPO appeared to decorate the glomerular capillaries and was closely adjacent to the staining of FH. In some areas of the glomerulus, co-localization of MPO and FH on endothelial cells was observed (Fig. 2). Nevertheless, glomerulus from normal controls and glomerulus from a patient with diabetic nephropathy demonstrated little staining of FH and no intraglomerular MPO with intact CD34+ endothelial cells. Staining with isotype controls is shown in supplementary Fig. S2, available at Rheumatology online. Fig. 1 View largeDownload slide Immunostaining of FH and MPO in NETs NETs were induced by incubating neutrophils isolated from healthy donors with serum from AAV patients. (A–C) NETs released from ANCA-activated neutrophils demonstrated positive staining of FH, co-localizing with the staining of MPO. (D and E) Isotype controls for staining antibodies were performed by incubating with normal goat IgG and mouse IgG1. Representative images from three independent experiments are shown. DAPI: 4′,6-diamidino-2-phenylindole. Fig. 1 View largeDownload slide Immunostaining of FH and MPO in NETs NETs were induced by incubating neutrophils isolated from healthy donors with serum from AAV patients. (A–C) NETs released from ANCA-activated neutrophils demonstrated positive staining of FH, co-localizing with the staining of MPO. (D and E) Isotype controls for staining antibodies were performed by incubating with normal goat IgG and mouse IgG1. Representative images from three independent experiments are shown. DAPI: 4′,6-diamidino-2-phenylindole. Fig. 2 View largeDownload slide Immunostaining of FH and MPO in renal specimens (A) Glomerulus from patients with AAV demonstrated positive staining of FH and MPO. MPO was closely adjacent to FH and there were areas showing co-localization of MPO and FH (arrow). (B and C) Glomerulus from normal controls and glomerulus from a patient with diabetic nephropathy demonstrated scanty staining of FH and no intraglomerular MPO. (D) FH was distributed along the capillary loops of endothelial cells in glomeruli of patients with AAV, co-localized with the staining of endothelial cells (CD34). (E and F) Glomerulus from normal controls and glomerulus from diabetic nephropathy showed scarce deposition of FH with intact CD34+ endothelial cells. Representative images from three independent experiments are shown. Fig. 2 View largeDownload slide Immunostaining of FH and MPO in renal specimens (A) Glomerulus from patients with AAV demonstrated positive staining of FH and MPO. MPO was closely adjacent to FH and there were areas showing co-localization of MPO and FH (arrow). (B and C) Glomerulus from normal controls and glomerulus from a patient with diabetic nephropathy demonstrated scanty staining of FH and no intraglomerular MPO. (D) FH was distributed along the capillary loops of endothelial cells in glomeruli of patients with AAV, co-localized with the staining of endothelial cells (CD34). (E and F) Glomerulus from normal controls and glomerulus from diabetic nephropathy showed scarce deposition of FH with intact CD34+ endothelial cells. Representative images from three independent experiments are shown. Binding between FH and MPO The binding between FH and MPO was determined by ELISA using a polyclonal antibody against FH. An increasing concentration of FH showed dose-dependent binding to coated MPO (Fig. 3A). To identify the binding sites for MPO on FH, we assessed the binding of recombinantly expressed FH fragments that span through the whole molecule to coated MPO. SCR1–4 of FH was identified as containing the major binding site for MPO (Fig. 3B). To rule out the possibility that FH was interacting with traces of DNA that still could not be completely isolated from MPO during purification, MPO was treated with 0–10 units of DNase-1 (Sigma) prior to FH incubation; no obvious difference in FH binding was detected (see supplementary Fig. S3, available at Rheumatology online). Considering that MPO serves as an autoantigen for ANCA in patients with MPO-ANCA-positive vasculitis, we assessed whether its interaction with FH is inhibited by ANCA. As expected, this interaction was not diminished in the presence of ANCA (see supplementary Fig. S4, available at Rheumatology online). Fig. 3 View largeDownload slide Analysis of MPO–FH interaction by microtitre plate binding assays and SPR (A) Dose-dependent binding of FH to MPO assayed by ELISA. A representative experiment of three performed is shown. (B) Mapping of the MPO–FH interaction sites by ELISA. Full-length FH and FH fragments were incubated with MPO-coated wells. The values represent mean (s.d.) derived from three independent experiments. (C) The SCR1–4 of FH was identified as the major binding site for MPO by SPR. Full-length FH and FH fragments were allowed to flow across an MPO immobilized chip; the signal obtained on a blank chip was subtracted. (D and E) SPR analysis of the binding kinetics of FH and SCR1–4 to the MPO immobilized chip. The affinity constant was 5.51 × 10−9 M and 5.95 × 10−9 M, respectively. (F and G) MPO bound to FH and SCR1–4 coupled chips with similar affinities, which was 1.28 × 10−9 M and 3.66 × 10−10 M, respectively. All SPR experiments were repeated twice independently. Fig. 3 View largeDownload slide Analysis of MPO–FH interaction by microtitre plate binding assays and SPR (A) Dose-dependent binding of FH to MPO assayed by ELISA. A representative experiment of three performed is shown. (B) Mapping of the MPO–FH interaction sites by ELISA. Full-length FH and FH fragments were incubated with MPO-coated wells. The values represent mean (s.d.) derived from three independent experiments. (C) The SCR1–4 of FH was identified as the major binding site for MPO by SPR. Full-length FH and FH fragments were allowed to flow across an MPO immobilized chip; the signal obtained on a blank chip was subtracted. (D and E) SPR analysis of the binding kinetics of FH and SCR1–4 to the MPO immobilized chip. The affinity constant was 5.51 × 10−9 M and 5.95 × 10−9 M, respectively. (F and G) MPO bound to FH and SCR1–4 coupled chips with similar affinities, which was 1.28 × 10−9 M and 3.66 × 10−10 M, respectively. All SPR experiments were repeated twice independently. To further confirm and characterize the interaction between MPO and FH, SPR was performed. The injection of FH on an MPO immobilized chip showed dose-dependent binding with an apparent KD of 5.51 × 10−9 M. At physiological pH and ionic strength, FH-MPO association and dissociation constants were 4.49 × 104/Ms (s.d. 0.03 × 104) and 2.47 × 10−4/s (s.d. 0.05 × 10−4), respectively. Using the same chip, SCR1–4 of FH was confirmed to contain the major binding site for MPO with an apparent KD of 5.95 × 10−9 M, which was comparable to the full-length FH (Fig. 3C–E). The calculated apparent association and dissociation constants were 9.68 × 104/Ms (s.d. 0.09 × 104) and 5.76 × 10−4/s (s.d. 0.03 × 10−4 ), respectively. Consistently, MPO bound to FH and SCR1–4 coupled chips with similar affinities, which was 1.28 × 10−9 M and 3.66 × 10−10 M, respectively (Fig. 3F and G). MPO inhibits the interaction of FH with C3b and the functional activity of FH in the regulation of C3b Interaction of FH with C3b is essential for the regulatory activities of FH, as it mediates the decay-accelerating and cofactor activities of FH. Therefore we first investigated whether the FH–C3b interaction could be altered in the presence of MPO. MPO inhibited the binding of FH to C3b in a dose-dependent manner. By incubating increasing concentrations of FH mixed with 2 μg/ml MPO in C3b-coated wells, we found the FH bound dose-dependently to C3b, but the interaction between FH and C3b was largely inhibited in the presence of MPO (P < 0.001; Fig. 4A and B). Then we tested the influence of the MPO–FH interaction on the decay-accelerating activity of FH. The results showed that FH accelerated the decay of C3 convertase (C3bBb), which was assembled on immobilized C3b, in a dose-dependent manner (Fig. 4C). In the presence 2 μg/ml MPO, the decay-accelerating activity of FH was significantly weakened [0.56 (s.d. 0.02) vs 0.70 (s.d. 0.04), P = 0.004 for FH at 10 μg/ml; 0.88 (s.d. 0.01) vs 1.02 (s.d. 0.04), P = 0.004 for FH at 5 μg/ml] (Fig. 4D). To explore whether MPO interferes with the cofactor activity of CFH upon C3b inactivation, the fluid phase cofactor activity of FH in the presence of MPO was assayed. MPO inhibited the cofactor activity of FH, as evidenced by the increased density of the uncleaved α′ 108 band and the decreased density of the cleaved α′ 43 band (Fig. 5A). Densitometric analyses of the relative intensity of the α′ 43 band showed that the fluid phase cofactor activity of FH was decreased by ∼40% in the presence of 2 μg/ml MPO [100% (s.d. 0.00) vs 58.98% (s.d. 5.13), P < 0.001] (Fig. 5B). We further found that cofactor activity of FH on human basement membrane extract MaxGel and HRGECs were also inhibited in the presence of 2 μg/ml MPO. Densitometric analyses showed that the cofactor activity of FH on such surfaces was decreased by ∼30% in the presence of 2 μg/ml MPO [100% (s.d. 0.00) vs 69.38% (s.d. 3.35), P < 0.001; 100% (s.d. 0.00) vs 70.79% (s.d. 2.28), P < 0.001, respectively] (Fig. 5C–F). Fig. 4 View largeDownload slide Influence of MPO on FH–C3b interaction and the decay-accelerating activity of FH (A) MPO inhibited the binding of FH to C3b in a dose-dependent manner (***P < 0.001, Student’s t test). The values represent mean (s.d.) derived from three independent experiments. (B) Binding of FH to coated C3b was inhibited in the presence of 2 μg/ml MPO (P < 0.001, two-way analysis of variance). Shown is the mean (s.d.) of three independent experiments. (C) Dose-dependent convertase decay-accelerating effects of FH; human serum albumin (HSA) was set as a control. (D) In the presence 2 μg/ml MPO, the decay-accelerating activity of FH was significantly weakened. The bars and error bars represent the mean (s.d.) of the remaining C3 convertase after decay by FH. Results are derived from three independent experiments. Fig. 4 View largeDownload slide Influence of MPO on FH–C3b interaction and the decay-accelerating activity of FH (A) MPO inhibited the binding of FH to C3b in a dose-dependent manner (***P < 0.001, Student’s t test). The values represent mean (s.d.) derived from three independent experiments. (B) Binding of FH to coated C3b was inhibited in the presence of 2 μg/ml MPO (P < 0.001, two-way analysis of variance). Shown is the mean (s.d.) of three independent experiments. (C) Dose-dependent convertase decay-accelerating effects of FH; human serum albumin (HSA) was set as a control. (D) In the presence 2 μg/ml MPO, the decay-accelerating activity of FH was significantly weakened. The bars and error bars represent the mean (s.d.) of the remaining C3 convertase after decay by FH. Results are derived from three independent experiments. Fig. 5 View largeDownload slide MPO inhibits the cofactor activity of FH (A) The fluid phase cofactor activity of FH for C3b cleavage was inhibited in the presence of MPO, as evidenced by the increased density of the uncleaved α′ 108 band and the decreased density of the cleaved α′ 43 band. A representative blot of three experiments is shown. (B) Densitometric analysis of the relative intensity of the cleavage fragment of C3b and the ratio of the α′ 43 band to the α′ 108 band. Data are presented as the mean (s.d.) of the percentage of C3b generation achieved with commercial FH (100%). The results of three independent experiments are shown. (C) The cofactor activity of FH on MaxGel was inhibited by MPO. (D) C3b cleavage was densitometrically quantified as described above. Results are derived from three independent experiments. (E) The cofactor activity of FH on HRGECs was inhibited by MPO. (F) Densitometric analysis of C3b proteolysis as described. Shown is the mean (s.d.) of three independent experiments (***P < 0.001). Fig. 5 View largeDownload slide MPO inhibits the cofactor activity of FH (A) The fluid phase cofactor activity of FH for C3b cleavage was inhibited in the presence of MPO, as evidenced by the increased density of the uncleaved α′ 108 band and the decreased density of the cleaved α′ 43 band. A representative blot of three experiments is shown. (B) Densitometric analysis of the relative intensity of the cleavage fragment of C3b and the ratio of the α′ 43 band to the α′ 108 band. Data are presented as the mean (s.d.) of the percentage of C3b generation achieved with commercial FH (100%). The results of three independent experiments are shown. (C) The cofactor activity of FH on MaxGel was inhibited by MPO. (D) C3b cleavage was densitometrically quantified as described above. Results are derived from three independent experiments. (E) The cofactor activity of FH on HRGECs was inhibited by MPO. (F) Densitometric analysis of C3b proteolysis as described. Shown is the mean (s.d.) of three independent experiments (***P < 0.001). Influence of MPO on the protective ability of FH against complement-mediated attack on sheep erythrocytes We further investigated whether MPO interferes with the protective ability of FH against complement-mediated haemolysis of sheep erythrocytes. Sheep erythrocytes lysis was induced in normal human plasma by mAb OX24, which inhibits the N-terminal complement regulatory domains. A dose-dependent inhibition of this lysis was achieved when FH was added (Fig. 6A). The addition of MPO did not interfere with the protective ability of FH against sheep erythrocytes lysis induced by mAb OX24 (Fig. 6B). However, levels of C3a and C5a in the supernatant were significantly higher when FH was pre-incubated with MPO (Fig. 6C and D). Fig. 6 View largeDownload slide Influence of MPO on the protective ability of FH against complement-mediated attack on sheep erythrocytes (A) Sheep erythrocytes lysis was induced by the FH mAb OX24 with 15% normal human serum. FH protects sheep erythrocytes from haemolysis induced by mAb OX24 in a dose-dependent manner. (B) MPO did not interfere with the protective ability of FH against sheep erythrocytes lysis induced by mAb OX24. (C and D) Pre-incubation of FH with MPO resulted in more C3a and C5a production in the supernatants. Data are mean (s.d.) from three independent experiments. Fig. 6 View largeDownload slide Influence of MPO on the protective ability of FH against complement-mediated attack on sheep erythrocytes (A) Sheep erythrocytes lysis was induced by the FH mAb OX24 with 15% normal human serum. FH protects sheep erythrocytes from haemolysis induced by mAb OX24 in a dose-dependent manner. (B) MPO did not interfere with the protective ability of FH against sheep erythrocytes lysis induced by mAb OX24. (C and D) Pre-incubation of FH with MPO resulted in more C3a and C5a production in the supernatants. Data are mean (s.d.) from three independent experiments. Discussion The interaction between neutrophils and activation of alternative complement pathway plays a pivotal role in the pathogenesis of AAV. An in vitro study showed that neutrophils activated by ANCA release factors capable of activating the complement system and generating C5a [3]. C5a further primes neutrophils for activation by ANCA [3, 9, 10], thus causing a self-amplification loop for ANCA-induced neutrophil activation. Among various neutrophil constituents that could be released upon activation, MPO was previously shown to be capable of activating the alternative complement pathway by interacting with properdin [11]. Our current study found that MPO interacts with the N-terminus of FH and inhibits the complement regulatory activity of FH, indicating a novel path for MPO in activating the complement system. Another interesting finding is that FH deposited in NETs and co-localized with MPO. By demonstrating that the complement regulatory activity of FH was inhibited by MPO, the current study further extended our previous finding that NETs released from ANCA-activated neutrophils are capable of activating the alternative complement pathway [20]. Moreover, by attracting FH binding, it might contribute to the decreased concentration of FH in the plasma of patients with AAV [18]. Based on our previous findings, decreased plasma levels of FH and deficient functional activities of FH were associated with disease activity and circulating levels of complement activation products in AAV [18, 19], indicating an important role of FH in disease development. Therefore our current finding indicates that the MPO–FH interaction may contribute to activation of the alternative complement pathway in AAV patients. Although, ANCA did not influence the binding of MPO to FH, we speculate that it would amplify the effects resulting from the MPO–FH interaction. By inhibiting FH, MPO contributes to the generation of more C5a, which would further prime neutrophils for activation by ANCA, resulting in more degranulation of MPO and thus causing a self-fuelling inflammatory amplification loop. In the renal specimens of patients with ANCA-associated glomerulonephritis, MPO was closely adjacent to FH localized to the endothelial surface of the capillary wall, which is consistent with the previous finding that MPO adheres to glomerular endothelial cells and is associated with the extent of endothelial injury in patients with ANCA-associated glomerulonephritis [28]. Although MPO is subendothelially concentrated [28, 29], there is considerable evidence demonstrating that MPO binds to endothelial cells and lasts for hours [29–31]. However, MPO would be transferred from the cell surface to cytoplasm, and possibly nuclear, through an internalization process by endothelial cells [30, 31]. It may account for the current observation that some areas of renal specimens from AAV patients showed co-localization of MPO and FH on endothelial cells, but in most areas of the glomeruli, MPO was subendothelially localized and closely adjacent to FH. Nevertheless, it could not exclude the possibility that the interaction of MPO with FH occurred on the glomerular endothelium. Moreover, given that MPO was able to mediate nitration of tyrosine residues during inflammatory processes [32, 33], and interestingly, nitration of tyrosines in factor H was recently reported to result in the loss of functional activities in interacting with C3b and cofactor activity for factor I–mediated cleavage of C3b [34], we speculate that MPO adhering to the glomerular endothelium might inhibit the negative regulation of alternative complement activation on endothelial cells. It extends the current understanding that MPO can mediate injury of glomerular capillary walls in AAV by direct oxidative damage and by triggering humoral and cellular immune response [35]. Although a significant inhibitory effect of MPO on FH-mediated inactivation of C3b on host cell surfaces was observed, no obvious effect can be detected in the haemolytic assays. However, we demonstrated that pre-incubation of FH with MPO resulted in more C3a and C5a production in the supernatants of haemolytic assays. Regarding the end product of complement activation, it is C5a, not C5b-9, that plays a critical role in the pathogenesis of AAV, as evidenced by the animal study [1, 8]. C5a not only primes neutrophils for activation by ANCA, but also amplifies the neutrophil–endothelial cell interactions on the surface of capillary walls [36]. Therefore we suggest that, by contributing to the generation of more C5a, MPO–FH interaction may contribute to the disease development of AAV. On the other hand, given that ceruloplasmin, the physiological inhibitor of MPO, exists in the serum, it is reasonable to speculate that it may present as a confounder for the result of the haemolysis assays. However, in patients with AAV, the inhibition of MPO by ceruloplasmin can be reversed by ANCA [37]. It would be of great interest to carry out in vivo studies to investigate the role of MPO on alternative complement activation and regulation in AAV. Overall, the effect of MPO’s interaction on FH cofactor activity and decay accelerating activity or C3a/C5a production is rather modest, which is a limitation of the current study. However, given the fact that the alternative complement pathway is characterized by self-amplification, it was suggested that the very nature of the complement system will amplify these small effects [25]. Considering that, along with AAV, alternative complement activation has been implicated in various neutrophil-mediated diseases, such as bacterial sepsis [38, 39], the interaction between MPO and FH and its effect on the complement regulatory activity of FH may not be limited in AAV. However, the biological significance of FH in bacterial sepsis is still controversial given that FH was previously reported to contribute to immune invasion of at least 10 pathogenic microbes [40–43]. Therefore it would be of great interest to investigate the role of the MPO–FH interaction in other diseases. In conclusion, MPO, which can be released from ANCA-stimulated neutrophils, binds to FH and inhibits the complement regulatory activity of FH. The MPO–FH interaction may participate in the pathogenesis of AAV by contributing to activation of the alternative complement pathway. Acknowledgements We are very grateful to Prof Ge Fu and Jing Wang from the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, for technical assistance on the surface plasmon resonance analysis. Funding: This work was supported by a grant from the National Key Research and Development Program (2016YFC0906102), three grants from the National Natural Science Fund (81400724, 81425008 and 81621092) and by a grant from the University of Michigan Health System and Peking University Health Sciences Center Joint Institute for Translational and Clinical Research. Disclosure statement: The authors have declared no conflicts of interest. 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RheumatologyOxford University Press

Published: Feb 19, 2018

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