Alternative Complement Pathway in the Pathogenesis of Disease Mediated by Anti-Neutrophil Cytoplasmic Autoantibodies

Clinical and experimental data indicate that anti-neutrophil cytoplasmic autoantibodies (ANCAs) cause glomerulonephritis and vasculitis. Here we report the first evidence that complement is an important mediator of ANCA disease. Transfer of anti-myeloperoxidase (MPO) IgG into wild-type mice or anti-MPO splenocytes into immune-deficient mice caused crescentic glomerulonephritis that could be completely blocked by complement depletion. The role of specific complement activation pathways was investigated using mice with knockout of the common pathway component C5, classic and lectin binding pathway component C4, and alternative pathway component factor B. After injection of anti-MPO IgG, C4−/− mice developed disease comparable with wild-type disease; however, C5−/− and factor B−/− mice developed no disease. To substantiate a role for complement in human ANCA disease, IgG was isolated from patients with myeloperoxidase ANCA (MPO-ANCA) or proteinase 3 ANCA (PR3-ANCA) and from controls. Incubation of MPO-ANCA or PR3-ANCA IgG with human neutrophils caused release of factors that activated complement. IgG from healthy controls did not produce this effect. The findings suggest that stimulation of neutrophils by ANCA causes release of factors that activate complement via the alternative pathway, thus initiating an inflammatory amplification loop that mediates the severe necrotizing inflammation of ANCA disease. Clinical and experimental data indicate that anti-neutrophil cytoplasmic autoantibodies (ANCAs) cause glomerulonephritis and vasculitis. Here we report the first evidence that complement is an important mediator of ANCA disease. Transfer of anti-myeloperoxidase (MPO) IgG into wild-type mice or anti-MPO splenocytes into immune-deficient mice caused crescentic glomerulonephritis that could be completely blocked by complement depletion. The role of specific complement activation pathways was investigated using mice with knockout of the common pathway component C5, classic and lectin binding pathway component C4, and alternative pathway component factor B. After injection of anti-MPO IgG, C4−/− mice developed disease comparable with wild-type disease; however, C5−/− and factor B−/− mice developed no disease. To substantiate a role for complement in human ANCA disease, IgG was isolated from patients with myeloperoxidase ANCA (MPO-ANCA) or proteinase 3 ANCA (PR3-ANCA) and from controls. Incubation of MPO-ANCA or PR3-ANCA IgG with human neutrophils caused release of factors that activated complement. IgG from healthy controls did not produce this effect. The findings suggest that stimulation of neutrophils by ANCA causes release of factors that activate complement via the alternative pathway, thus initiating an inflammatory amplification loop that mediates the severe necrotizing inflammation of ANCA disease. Anti-neutrophil cytoplasmic autoantibodies (ANCA) are specific for proteins in the cytoplasm of neutrophils and monocytes. The major target antigens in patients with vasculitis and glomerulonephritis are myeloperoxidase (MPO) and proteinase 3 (PR3). ANCAs occur in greater than 80% of patients with active untreated Wegener's granulomatosis, microscopic polyangiitis, and pauci-immune crescentic glomerulonephritis.1Jennette JC Xiao H Falk RJ The pathogenesis of vascular inflammation by antineutrophil cytoplasmic antibodies.J Am Soc Nephrol. 2006; 17: 1235-1242Crossref PubMed Scopus (165) Google Scholar There is compelling clinical and experimental evidence that ANCA IgG causes ANCA-associated vasculitis and glomerulonephritis. The strongest clinical evidence for causation is the observation that a newborn child developed glomerulonephritis and pulmonary hemorrhage shortly after delivery from a mother with MPO-ANCA-associated microscopic polyangiitis, apparently caused by transplacental transfer of ANCA IgG.2Bansal PJ Tobin MC Neonatal microscopic polyangiitis secondary to transfer of maternal myeloperoxidase-antineutrophil cytoplasmic antibody resulting in neonatal pulmonary hemorrhage and renal involvement.Ann Allergy Asthma Immunology. 2004; 93: 398-401Abstract Full Text PDF PubMed Scopus (173) Google Scholar, 3Schlieben DJ Korbet SM Kimura RE Schwartz MM Lewis EJ Pulmonary-renal syndrome in a newborn with placental transmission of ANCAs.Am J Kidney Dis. 2005; 45: 758-761Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar Two compelling animal models of ANCA vasculitis and glomerulonephritis have been described by two different research groups.4Xiao H Heeringa P Hu P Liu Z Zhao M Aratani Y Maeda N Falk RJ Jennette JC Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice.J Clin Invest. 2002; 110: 955-963Crossref PubMed Scopus (986) Google Scholar, 5Little MA Smyth CL Yadav R Ambrose L Cook HT Nourshargh S Pusey CD Anti-neutrophil cytoplasm antibodies directed against myeloperoxidase augment leukocyte-microvascular interactions in vivo.Blood. 2005; 106: 2050-2058Crossref PubMed Scopus (238) Google Scholar Xiao and colleagues4Xiao H Heeringa P Hu P Liu Z Zhao M Aratani Y Maeda N Falk RJ Jennette JC Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice.J Clin Invest. 2002; 110: 955-963Crossref PubMed Scopus (986) Google Scholar induced glomerulonephritis and systemic vasculitis by intravenous injection of either anti-MPO IgG or anti-MPO splenocytes derived from MPO knockout mice immunized with murine MPO. Induction of glomerulonephritis by anti-MPO IgG in this model is enhanced by cytokines6Huugen D Xiao H van Esch A Falk RJ Peutz-Kootstra CJ Buurman WA Tervaert JW Jennette JC Heeringa P Aggravation of anti-myeloperoxidase antibody-induced glomerulonephritis by bacterial lipopolysaccharide: role of tumor necrosis factor-alpha.Am J Pathol. 2005; 167: 47-58Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar and requires neutrophils.7Xiao H Heeringa P Liu Z Huugen D Hu P Maeda N Falk RJ Jennette JC The role of neutrophils in the induction of glomerulonephritis by anti-myeloperoxidase antibodies.Am J Pathol. 2005; 167: 39-45Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar Little and colleagues5Little MA Smyth CL Yadav R Ambrose L Cook HT Nourshargh S Pusey CD Anti-neutrophil cytoplasm antibodies directed against myeloperoxidase augment leukocyte-microvascular interactions in vivo.Blood. 2005; 106: 2050-2058Crossref PubMed Scopus (238) Google Scholar immunized rats with human MPO, resulting in the production of antibodies that cross reacted with rat MPO and caused vasculitis and glomerulonephritis. The pathogenic effects of these anti-MPO antibodies were augmented by cytokines.Numerous in vitro studies indicate that ANCA IgG can activate neutrophils and cause them to release proinflammatory factors. The expression of ANCA antigens (MPO and PR3) at the surface of neutrophils where they are accessible to interact with ANCA IgG is enhanced by proinflammatory cytokines, such as tumor necrosis factor (TNF)-α.8Falk RJ Terrell R Charles LA Jennette JC Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals.Proc Natl Acad Sci USA. 1990; 87: 4115-4119Crossref PubMed Scopus (1100) Google Scholar, 9Charles LA Caldas ML Falk RJ Terrell RS Jennette JC Antibodies against granule proteins activate neutrophils in vitro.J Leuk Biol. 1991; 50: 539-546PubMed Google Scholar Incubation of TNF-α-primed neutrophils with ANCA IgG induces full activation with release of lytic and toxic granule enzymes and reactive oxygen species.8Falk RJ Terrell R Charles LA Jennette JC Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals.Proc Natl Acad Sci USA. 1990; 87: 4115-4119Crossref PubMed Scopus (1100) Google Scholar, 9Charles LA Caldas ML Falk RJ Terrell RS Jennette JC Antibodies against granule proteins activate neutrophils in vitro.J Leuk Biol. 1991; 50: 539-546PubMed Google Scholar Interaction of ANCA IgG with ANCA antigens in the microenvironment of neutrophils causes activation through both Fc receptor engagement and Fab′2 binding.10Porges AJ Redecha PB Kimberly WT Csernok E Gross WL Kimberly RP Anti-neutrophil cytoplasmic antibodies engage and activate human neutrophils via Fc gamma RIIa.J Immunol. 1994; 153: 1271-1280PubMed Google Scholar, 11Mulder AH Heeringa P Brouwer E Limburg PC Kallenberg CG Activation of granulocytes by anti-neutrophil cytoplasmic antibodies (ANCA): a Fc gamma RII-dependent process.Clin Exp Immunol. 1994; 98: 270-278Crossref PubMed Scopus (233) Google Scholar, 12Kettritz R Jennette JC Falk RJ Crosslinking of ANCA-antigens stimulates superoxide release by human neutrophils.J Am Soc Nephrol. 1997; 8: 386-394PubMed Google Scholar, 13Williams JM Ben-Smith A Hewins P Dove SK Hughes P McEwan R Wakelam MJ Savage CO Activation of the G(i) heterotrimeric G protein by ANCA IgG F(ab′)2 fragments is necessary but not sufficient to stimulate the recruitment of those downstream mediators used by intact ANCA IgG.J Am Soc Nephrol. 2003; 14: 661-669Crossref PubMed Scopus (80) Google Scholar Activation of neutrophil by ANCA IgG in the presence of cultured endothelial cells results in neutrophil adherence,14Radford DJ Savage CO Nash GB Treatment of rolling neutrophils with antineutrophil cytoplasmic antibodies causes conversion to firm integrin-mediated adhesion.Arthritis Rheum. 2000; 43: 1337-1345Crossref PubMed Scopus (113) Google Scholar neutrophil transmigration,15Radford DJ Luu NT Hewins P Nash GB Savage CO Antineutrophil cytoplasmic antibodies stabilize adhesion and promote migration of flowing neutrophils on endothelial cells.Arthritis Rheum. 2001; 44: 2851-2861Crossref PubMed Scopus (119) Google Scholar and endothelial cell death.16Ewert BH Jennette JC Falk RJ Anti-myeloperoxidase antibodies stimulate neutrophils to damage human endothelial cells.Kidney Int. 1992; 41: 375-383Crossref PubMed Scopus (226) Google Scholar, 17Savage CO Pottinger BE Gaskin G Pusey CD Pearson JD Autoantibodies developing to myeloperoxidase and proteinase 3 in systemic vasculitis stimulate neutrophil cytotoxicity toward cultured endothelial cells.Am J Pathol. 1992; 141: 335-342PubMed Google Scholar Little and colleagues5Little MA Smyth CL Yadav R Ambrose L Cook HT Nourshargh S Pusey CD Anti-neutrophil cytoplasm antibodies directed against myeloperoxidase augment leukocyte-microvascular interactions in vivo.Blood. 2005; 106: 2050-2058Crossref PubMed Scopus (238) Google Scholar have documented this process in vivo using their rat model to show by intravital microscopy that leukocytes activated with MPO-ANCA IgG adhere to and injure the microvasculature.Before the observations reported in this article, a role for complement in the pathogenesis of ANCA-induced inflammation has not been suspected. This is in part because there is less complement deposition in vessel walls in ANCA vasculitis and glomerulonephritis as compared with the substantial complement deposition that is observed with immune complex disease and anti-glomerular basement membrane disease.18Jennette JC Wilkman AS Falk RJ Anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis and vasculitis.Am J Pathol. 1989; 135: 921-930PubMed Google Scholar, 19Jennette JC Implications for pathogenesis of patterns of injury in small and medium-sized vessel vasculitis.Cleveland Clinic J Med. 2002; 69: SII33-SII38Crossref PubMed Google Scholar However, an important mediator of vascular inflammation does not have to be present in vessel walls at high concentrations. For example, there is a paucity of IgG in the vascular lesions of ANCA vasculitis and glomerulonephritis, yet, as reviewed earlier, there is compelling evidence that ANCA IgG is the primary pathogenic factor causing these inflammatory lesions. ANCA disease is not associated with hypocomplementemia; however, this is not a sensitive indicator of complement involvement because certain forms of glomerulonephritis and vasculitis that have substantial vascular deposits of complement do not have hypocomplementemia, such as Henoch-Schönlein purpura and anti-GBM disease. In addition, complement activation has been identified as a major mediator of injury and inflammation in a variety of diseases in which there is little or no complement localization at the site of injury, for example, complement activation, probably through the alternative pathway, is an important mediator in ischemia reperfusion injury.20Riedemann NC Ward PA Complement in ischemia reperfusion injury.Am J Pathol. 2003; 162: 363-367Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar The complement system can be activated through three different pathways: classic, lectin, and alternative.21Seelen MA Roos A Daha MR Role of complement in innate and autoimmunity.J Nephrol. 2005; 18: 642-653PubMed Google Scholar, 22Agrawal A CRP after 2004.Mol Immunol. 2005; 42: 927-930Crossref PubMed Scopus (78) Google Scholar, 23Thurman JM Holers VM The central role of the alternative complement pathway in human disease.J Immunol. 2006; 176: 1305-1310PubMed Scopus (297) Google Scholar Among the many factors that can activate complement are mediators released by activated neutrophils.24Shingu M Nonaka S Nishimukai H Nobunaga M Kitamura H Tomo-Oka K Activation of complement in normal serum by hydrogen peroxide and hydrogen peroxide-related oxygen radicals produced by activated neutrophils.Clin Exp Immunol. 1992; 90: 72-78Crossref PubMed Scopus (67) Google Scholar, 25Vogt W Complement activation by myeloperoxidase products released from stimulated human polymorphonuclear leukocytes.Immunobiology. 1996; 195: 334-346Crossref PubMed Scopus (53) Google Scholar, 26Venge P Olsson I Cationic proteins of human granulocytes. VI. Effects on the complement system and mediation of chemotactic activity.J Immunol. 1975; 115: 1505-1508PubMed Google Scholar, 27Wirthmueller U Dewald B Thelen M Schafer MK Stover C Whaley K North J Eggleton P Reid KB Schwaeble WJ Properdin, a positive regulator of complement activation, is released from secondary granules of stimulated peripheral blood neutrophils.J Immunol. 1997; 158: 4444-4451PubMed Google Scholar Based on the observations reported herein, we hypothesize that ANCA-induced activation of neutrophils results in the release of factors that activate the alternative complement pathway amplification loop, which in turn augments recruitment and activation of more neutrophils, resulting in the severe necrotizing leukocytoclastic inflammation that is so characteristic of acute ANCA disease.Materials and MethodsMiceMPO knockout (Mpo−/−) mice initially generated by Aratani and colleagues28Aratani Y Koyama H Nyui S Suzuki K Kura F Maeda N Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase.Infect Immun. 1999; 67: 1828-1836Crossref PubMed Google Scholar were maintained by the University of North Carolina Division of Laboratory Animal Medicine. Mpo−/− mice (8 to 10 weeks old) were used for immunization and as donors of anti-MPO antibodies and splenocytes. Rag2 mice were purchased from Taconic Farms (Germantown, NY). Factor B−/− mice that were initially generated by Matsumoto and colleagues29Matsumoto M Fukuda W Circolo A Goellner J Strauss-Schoenberger J Wang X Fujita S Hidvegi T Chaplin DD Colten HR Abrogation of the alternative complement pathway by targeted deletion of murine factor B.Proc Natl Acad Sci USA. 1997; 94: 8720-8725Crossref PubMed Scopus (174) Google Scholar were kindly provided by Dr. Zhi Liu, Department of Dermatology, University of North Carolina, Chapel Hill, NC. Age- and sex-matched wild-type (WT) B6 controls, C4−/− mice, and C5−/− mice were purchased from Jackson Laboratory (Bar Harbor, ME). All complement-deficient mice had a C57BL/6 (B6) background. Rag2−/− mice, 10 to 12 weeks old, were used as recipients for adoptive splenocyte transfer experiments. Factor B−/−, C4−/−, C5−/−, and WT B6 mice, 9 to 10 weeks old, were used for IgG transfer experiments. All animal experiments were approved by the Animal Studies Committee of the University of North Carolina at Chapel Hill, School of Medicine, and were used in accordance with National Institutes of Health guidelines.Kinetics of C3 Depletion by Cobra Venom Factor (CVF)To determine the kinetics of C3 depletion by CVF, WT B6 mice (n = 6) were injected intraperitoneally with a single dose of 30 μg of CVF (Sigma-Aldrich, St. Louis, MO) in 0.3 ml of phosphate-buffered saline (PBS). Control mice (n = 6) received 0.3 ml of PBS. Serum C3 levels was measured on days 0, 1, 3, 5, 7, 9, 11, and 13 after injection of CVF by C3 antibody capture enzyme-linked immunosorbent assay (ELISA) using F(ab′)2 fragments of goat anti-mouse C3 as capture antibody and peroxidase-conjugated goat anti-mouse C3 as the second antibody (Cappel, Organon Teknika Corporate, West Chester, PA), according to the manufacturer's protocol. C3 levels monitored by ELISA were markedly reduced with the lowest level within 24 hours after injection and remained at this low level for up to 7 days. Thereafter, C3 returned toward normal and reached the normal level at approximately day 11. Control mice injected with the same volume of PBS exhibited normal levels of circulating C3 (Figure 1).Preparation of Nephritogenic Mouse Anti-Murine MPO Splenocytes and AntibodiesPurification of mouse MPO and the immunization of Mpo−/− mice were performed as previously described.4Xiao H Heeringa P Hu P Liu Z Zhao M Aratani Y Maeda N Falk RJ Jennette JC Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice.J Clin Invest. 2002; 110: 955-963Crossref PubMed Scopus (986) Google Scholar In brief, mouse MPO was purified from WEHI-3 cells by Dounce homogenization, Concanavalin A affinity chromatography, ion exchange, and gel filtration chromatography. Mpo−/− mice were immunized intraperitoneally with 10 μg of purified murine MPO or bovine serum albumin in complete Freund's adjuvant and subsequently boosted with antigen in incomplete Freund's adjuvant. Development of antibodies was monitored by ELISA and anti-MPO antibody reactivity with neutrophils was confirmed by indirect immunofluorescence microscopy on murine neutrophils. The IgG fraction was isolated from serum by 50% ammonium sulfate precipitation and protein G affinity chromatography. Splenocytes were isolated from immunized and nonimmunized Mpo−/− mice by disrupting the spleens into cold RPMI 1640 medium and then washing twice with RPMI 1640. Red blood cells were removed with lysis buffer (Sigma-Aldrich) followed by washing with RPMI 1640 and final suspension in sterile PBS.Induction of Glomerulonephritis with Anti-MPO IgG and Anti-MPO SplenocytesInduction of experimental necrotizing and crescentic glomerulonephritis (NCGN) with anti-MPO IgG in WT mice or anti-MPO splenocytes in Rag2−/− immune-deficient mice was performed as previously described.4Xiao H Heeringa P Hu P Liu Z Zhao M Aratani Y Maeda N Falk RJ Jennette JC Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice.J Clin Invest. 2002; 110: 955-963Crossref PubMed Scopus (986) Google Scholar Briefly, in antibody transfer experiments, mice were injected intravenously with 50 μg/g body weight (BW) of anti-MPO or control IgG in PBS and sacrificed on day 6. In splenocyte transfer experiments, Rag2−/− mice were injected intravenously with 5 × 107 anti-MPO or control splenocytes in 0.5 ml of PBS and sacrificed on day 13. At the point of sacrifice, serum and urine samples were collected to evaluate kidney disease, and serum samples also were collected for serological assays of antibody and complement activity.In IgG transfer CVF complement depletion experiments, WT and control mice received intravenously 50 μg/g BW of anti-MPO IgG 4 hours after injection of one dose of CVF or PBS, respectively, and were sacrificed on day 6. The effect of CVF on circulating complement was determined by C3 antibody capture ELISA. The urine, serum, and kidney tissue samples were collected for further evaluation as below.In splenocyte transfer CVF complement depletion experiments, Rag2−/− mice were injected intraperitoneally with 30 μg of CVF in 0.3 ml of PBS on days 0 and 6, and control mice received 0.3 ml of PBS on days 0 and 6. Both experimental and control mice received 5 × 107 anti-MPO splenocytes by intravenous injection 4 hours after receiving the first dose of CVF or PBS. The mice were sacrificed on day 13.In IgG transfer experiments in complement knockout mice, C5−/−, C4−/−, fB−/−, or WT mice were injected intravenously with 50 μg/g BW of anti-MPO IgG and sacrificed on day 6. The urine, serum, and kidney tissues were collected for evaluation.Functional Evaluation of Renal InjuryMice were placed in metabolic cages 1 day before sacrifice and urine was collected for 12 hours. Urine was tested by dipstick (Roche Diagnostics Corp., Indianapolis, IN) for hematuria and leukocyturia. The extent of hematuria and leukocyturia was expressed as the mean on a scale of 0 (none) to 4 (severe). Albuminuria was determined by a mouse albumin ELISA quantitation kit (Bethyl Laboratories Inc., Montgomery, TX). Using the mean ± 2 SDs for the reference range established in 305 healthy B6 mice, abnormal hematuria was set at >0.9 and leukocyturia >0.2.7Xiao H Heeringa P Liu Z Huugen D Hu P Maeda N Falk RJ Jennette JC The role of neutrophils in the induction of glomerulonephritis by anti-myeloperoxidase antibodies.Am J Pathol. 2005; 167: 39-45Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar Serum samples were collected at the time of sacrifice. Serum creatinine and blood urea nitrogen were measured using a Johnson & Johnson Ortho-Clinical Diagnostics VITROS 250 (Raritan, NJ).Pathological Evaluation of Renal InjurySamples of kidney tissue were collected at the time of sacrifice and fixed in 10% formalin, embedded in paraffin using routine protocols, sectioned at 4-μm, stained with hematoxylin and eosin and periodic acid Schiff, and evaluated by light microscopy. The extent of glomerular crescents and necrosis were expressed as the mean percentage of glomeruli with crescents and necrosis in each animal.For immunofluorescence microscopy to detect glomerular localization of immune determinants, 4-μm frozen sections were stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Molecular Probes Invitrogen, Carlsbad, CA), IgM, IgA, and MPO (ICN/Cappel, Aurora, OH), respectively. Deposition of complement C3 was visualized with fluorescein isothiocyanate-conjugated goat anti-mouse C3 (ICN/Cappel). Immunofluorescence microscopy staining of glomeruli for IgG, IgM, IgA, C3, and MPO was expressed as the mean intensity of staining on a scale of 0 (none) to 4 (severe).For immunohistochemistry to detect renal leukocytes infiltration, 4-μm frozen sections were stained with rat anti-mouse neutrophil Gr-1 (clone RB6-8C5; BD Pharmingen, Franklin Lakes, NJ) and rat anti-mouse monocytes/macrophages CD68 antibodies (clone FA11; Serotec, Raleigh, NC). Rat antibody binding was detected using peroxidase-conjugated secondary rabbit anti-rat IgG and tertiary goat anti-rabbit IgG antibodies (DAKO, Carpinteria, CA). Staining was visualized with 3-amino-9-ethylcarbazole and hydrogen peroxide. Sections were counterstained with hematoxylin. Leukocyte localization was expressed as the mean number of leukocytes per cross-section of glomeruli based on evaluating an average of 75 glomeruli per specimen (range, 60 to 90 glomeruli).Measurement of Complement Activation by Factors Released by ANCA-Stimulated Human NeutrophilsIgG from healthy controls (n = 8), MPO-ANCA patients (n = 6), or PR3-ANCA patients (n = 8) was prepared from plasma by ammonium sulfate precipitation and protein G affinity column chromatography. IgG preparation were passed over AffinityPak endotoxin removal columns (Pierce, Rockford, IL) before use. The plasma was obtained during plasma exchange on ANCA-positive patients with active, biopsy-proven necrotizing and crescentic glomerulonephritis. The patients also were receiving immunosuppressive treatment. Neutrophils were isolated from healthy volunteers using Ficoll sedimentation. Neutrophils from a single donor were used for each experimental run that compared the effect of control IgG to various experimental preparations. Autologous serum was obtained from the same donors as the neutrophils. In preparation to determination of the ability of IgG to stimulate neutrophils to release complement-activating factors, neutrophils were primed for 15 minutes at 37°C with 2 ng/ml TNF-α. The primed neutrophils then were incubated with 200 μg/ml of either control IgG, MPO-ANCA IgG, or PR3-ANCA IgG for 15 minutes at 37°C. Additional controls included reaction mixtures that contained control IgG, MPO-ANCA IgG, or PR3-ANCA IgG but no neutrophils (to determine whether the IgG alone could activate complement) and reaction mixtures that contained TNF-primed neutrophils but no immunoglobulin (to determine whether primed neutrophils alone could activate complement). The reaction mixtures were centrifuged at 300 g for 5 minutes at 4°C. A 100-μl aliquot of supernatant was added to precooled tubes containing 100 μl of autologous serum and incubated for 1 hour at 37°C. The reaction was stopped by adding ethylenediaminetetraacetic acid to a final concentration of 10 mmol/L. The supernatants were stored frozen at −70°C until assayed for C3a. C3a, which is a sensitive marker of complement activation, was assayed in the supernatant using the Quidel C3a EIA kit (Quidel Corp., San Diego, CA) by the manufacturer's instructions. All determinations were made in duplicate. Two or more control IgG samples were included with each assay, and results for individual control and experimental samples were expressed as a percentage of the result of the control IgG mean.ResultsComplement Is Required for Induction of Glomerulonephritis by Anti-MPO IgGPauci-immune NCGN that closely mimics human ANCA glomerulonephritis is induced in mice by a single injection of anti-MPO IgG derived from Mpo−/− mice that have been immunized with mouse MPO.4Xiao H Heeringa P Hu P Liu Z Zhao M Aratani Y Maeda N Falk RJ Jennette JC Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice.J Clin Invest. 2002; 110: 955-963Crossref PubMed Scopus (986) Google Scholar, 6Huugen D Xiao H van Esch A Falk RJ Peutz-Kootstra CJ Buurman WA Tervaert JW Jennette JC Heeringa P Aggravation of anti-myeloperoxidase antibody-induced glomerulonephritis by bacterial lipopolysaccharide: role of tumor necrosis factor-alpha.Am J Pathol. 2005; 167: 47-58Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 7Xiao H Heeringa P Liu Z Huugen D Hu P Maeda N Falk RJ Jennette JC The role of neutrophils in the induction of glomerulonephritis by anti-myeloperoxidase antibodies.Am J Pathol. 2005; 167: 39-45Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar By day 6, all mice that receive anti-MPO IgG develop segmental fibrinoid necrosis and crescents in ∼5 to 15% of glomeruli. Immunohistology demonstrates only mild staining for immunoglobulins and complement. The effect of complement depletion on the induction of NCGN in this model was evaluated. WT B6 mice were pretreated with (n = 7) or without (n = 7) a single intraperitoneal injection of CVF 4 hours before injection of anti-MPO IgG. Six days after injecting anti-MPO IgG, all mice that did not have complement depletion developed albuminuria, leukocyturia, and hematuria whereas the mice that received CVF stayed within the reference range (Figure 2, A and B). At the time of sacrifice, mice that received CVF had the same level of circulating anti-MPO IgG (Figure 2C) as the mice that did not receive CVF; however, as expected, they had decreased serum C3 levels (Figure 2D). All of the mice that received anti-MPO IgG without CVF developed NCGN with neutrophil and macrophage infiltration and low-level glomerular IgG and C3 deposition (Figure 3, A–C; and Table 1, Table 2). In contrast, none of the complement-depleted mice injected with the same dose of anti-MPO IgG developed glomerular necrosis or crescents (Figure 3, D–F; and Table 1, Table 2). The mice that did not receive CVF had a paucity of staining for C3 (0.5+) and mice that received CVF had no staining for C3 (Table 2).Figure 2Mice depleted of complement are resistant to anti-MPO IgG-induced experimental glomerulonephritis. WT mice were pretreated with a single dose of CVF or PBS (n = 7 for each group). Four hours later, the mice received anti-MPO IgG (50 μg/g body weight). A and B: Urine analysis showed that untreated mice and complement-depleted mice that received anti-MPO IgG had no urine abnormalities, whereas noncomplement-depleted mice that received anti-MPO IgG had albuminuria, hematuria, and leukocyturia. C and D: ELISA analysis demonstrated the same circulating anti-MPO IgG titers in noncomplement-depleted and complement-depleted mice (C); and low circulating C3 in complement-depleted mice (D). Extent of hematuria and leukocyturia is expressed as the mean on a scale of 0 (none) to 4 (severe). *P < 0.05 and #P < 0.001 versus untreated WT. **P < 0.004 compared with noncomplement-depleted mice. Bars represent the mean ± SD.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Renal tissue was examined 6 days after mice received anti-MPO IgG. WT mice that received pathogenic IgG without complement depletion developed focal glomerular necrosis (l