C5a Receptor Mediates Neutrophil Activation and ANCA-Induced Glomerulonephritis
Adrian Schreiber*,
Hong Xiao,
J. Charles Jennette,
Wolfgang Schneider*,
Friedrich C. Luft* and
Ralph Kettritz*
* Medical Faculty of the Charité, Department of Nephrology and Hypertension, Franz Volhard Clinic at the Max Delbrück Center for Molecular Medicine, HELIOS-Klinikum-Berlin, Berlin, Germany; and Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina
Correspondence: Dr. Adrian Schreiber, Schwanebecker Chaussee 50, 13125 Berlin, Germany. Phone: +49-30-9401-52800; Fax: +49-30-9401-52809; E-mail: adrian.schreiber{at}gmx.de
Received for publication May 15, 2008.
Accepted for publication September 5, 2008.
Anti-neutrophil cytoplasmic autoantibody (ANCA)-induced necrotizingcrescentic glomerulonephritis (NCGN) requires complement participationin its pathogenesis. We tested the hypothesis that the anaphylatoxinC5a is pivotal to disease induction via the neutrophil C5a receptor(C5aR). Supernatants from ANCA-activated neutrophils activatedthe complement cascade in normal serum, producing C5a. Thisconditioned serum primed neutrophils for ANCA-induced respiratoryburst; neutrophil C5aR blockade abrogated this priming, butC3aR blockade did not. Furthermore, recombinant C5a but notC3a dosage-dependently primed neutrophils for ANCA-induced respiratoryburst. To test the role of C5aR in a model of NCGN, we immunizedmyeloperoxidase-deficient mice with myeloperoxidase, irradiatedthem, and transplanted bone marrow from wild-type mice or C5aR-deficientmice into them. All mice that received wild-type marrow (sixof six) but only one of eight mice that received C5aR-deficientmarrow developed NCGN (P < 0.05). Albuminuria and neutrophilinflux into glomeruli were also significantly attenuated inthe mice that received C5aR-deficient marrow (P < 0.05).In summary, C5a and the neutrophil C5aR may compose an amplificationloop for ANCA-mediated neutrophil activation. The C5aR may providea new therapeutic target for ANCA-induced necrotizing crescenticglomerulonephritis.
Anti-neutrophil cytoplasmic antibodies (ANCA) are common indiseases that feature pauci-immune necrotizing crescentic glomerulonephritis(NCGN).1 ANCA are directed against either myeloperoxidase (MPO)or proteinase 3 (PR3). ANCA activate neutrophils, resultingin respiratory burst, degranulation, and adhesion molecule upregulation.2–6Recent animal models proved the pathogenicity of MPO-ANCA forNCGN induction in vivo.7,8 In one model, transfer of anti-MPOIgG into healthy wild-type (WT) mice resulted in NCGN that closelyresembled human ANCA disease.7 We recently modified this model,using a bone marrow (BM) transplantation approach. We used MPO-deficientmice that were immunized with murine MPO, subsequently wereirradiated, and received a transplant of WT BM. We then observeda longer lasting NCGN compared with other mouse models.9
The complement system consists of numerous plasma proteins,membrane-bound and circulating complement regulators, and complementreceptors expressed on various cell types. Three different complementactivation pathways are known: The classical, the lectin, andthe alternative pathway. Independent of the initiating trigger,all three lead to C3 convertase formation that cleaves C3 toC3b and C3a. C3b contributes to the formation of C5 convertase,which cleaves C5 to C5a and C5b. C5b mediates the formationof the C5b-9 terminal membrane attack complex (MAC). The MACresults in lytic and sublytic injury of targeted membrane structures.Independent of MAC effects, the small cleavage products C3a,C4a, and C5a are anaphylatoxins that function as potent inflammatorymediators via specific receptors on both myeloid and nonmyeloidcells.
Complement participation in human ANCA disease was not expected,because human ANCA-NCGN shows only a few glomerular C3 and C4deposits and overt systemic complement consumption with decreasedserum C3 and C4 levels do not occur; however, two differentanimal studies established the importance of the complementsystem in ANCA disease.10,11 We found recently that the alternativecomplement pathway is fundamental; mice with factor B or C3deficiency were completely protected from ANCA-induced NCGN.10We also observed that factors released from ANCA-activated neutrophilsinitiated the complement cascade, resulting in the generationof C3a. Because neutrophils express C3a receptor (C3aR) andC5aR, we tested the hypothesis that C3a or C5a is pivotal inANCA-induced NCGN. Our data establish the importance of C5aand its neutrophil receptor. C5aR blockade may provide a noveltherapeutic target.
ANCA-Stimulated Neutrophils Activate Complement and Generate C5a in Normal Serum
We first studied whether ANCA-stimulated neutrophils cause anaphylatoxinC5a generation in normal human serum. We primed neutrophilswith TNF- and subsequently stimulated them with human MPO-ANCAor PR3-ANCA IgG, resulting in neutrophil activation. We documentedactivation by estimating the degranulation response, measuredas β-glucoronidase release and respiratory burst, measuredas superoxide release. Degranulation was 5.7 ± 0.7% inunstimulated cells, 4.9 ± 2.0% in control IgG-treatedcells, 15.0 ± 5.4% after treatment with MPO-ANCA, and27.1 ± 13.0% (n = 4; P < 0.05) with PR3-ANCA. Superoxideproduction was 2.0 ± 0.6 nmol/106 neutrophils in unstimulatedcells, 4.0 ± 0.6 in control IgG-treated cells, 19.0 ±2.5 after treatment with MPO-ANCA, and 20 ± 3.0 aftertreatment with PR3-ANCA (n = 4; P < 0.05). We then co-incubatedthe supernatants for 15 min with an equal volume of normal serum(from here on called conditioned serum). Figure 1 shows C5ageneration in the conditioned sera as measured by ELISA. Thesedata show that activation of TNF-–primed neutrophils withhuman ANCA preparation resulted in the release of factors intothe supernatant that led to C5a generation in normal serum.In contrast, co-incubation of normal serum with buffer control,supernatants of TNF-–primed neutrophils that were notstimulated with ANCA, or supernatants of TNF-–primed neutrophilsstimulated with healthy IgG, respectively, did not lead to C5ageneration.
Figure 1. Normal TNF-–primed neutrophils were stimulated for 45 min with buffer control (buffer), 125 µg/ml normal healthy IgG (Ctrl IgG), or 125 µg/ml ANCA IgG (five independent experiments using MPO-ANCA in two experiments and PR3-ANCA in three experiments). Supernatants from the cell suspensions were incubated for 15 min with normal serum, and C5a generation was measured by a C5a-specific ELISA. Data are means ± SEM (given as ratio to buffer-incubated normal serum [–], which was set at baseline to 100%). **P < 0.01.
C3a- and C5a-Conditioned Serum Increases Neutrophil PR3 Membrane Expression
We showed in our previous study that C3a was also generatedin conditioned sera.10 In this study, we tested whether theC3a- and C5a-containing conditioned sera would affect ANCA antigenmembrane expression. Increases in membrane-PR3 expression aremuch stronger during neutrophil priming compared with MPO. Weincubated neutrophils with conditioned serum, and membrane-PR3expression was determined by flow cytometry. Figure 2 showsthat neutrophil incubation with conditioned serum obtained byincubating normal serum with supernatant from ANCA-treated neutrophilsbut not from neutrophils treated with normal IgG resulted inupregulation of membrane-PR3 expression. We repeated the experimentsusing hirudin-treated plasma instead of serum and recorded similareffects. Membrane-PR3 expression in neutrophils treated withplasma that was co-incubated with supernatants from TNF-–primedneutrophils stimulated with control IgG was set 100%. When plasmawas co-incubated with supernatants from TNF-–primed neutrophilsstimulated with ANCA IgG, membrane-PR3 increased to 154 ±9.0% (n = 3). These results establish an important biologiceffect of conditioned serum with implications for ANCA-inducedneutrophil activation.
Figure 2. Normal TNF-–primed neutrophils were stimulated for 45 min with buffer control (buffer), 125 µg/ml normal IgG (Ctrl IgG), or 125 µg/ml ANCA IgG (four independent experiments using MPO-ANCA in two experiments and PR3-ANCA in two experiments). Supernatants of these were incubated for 15 min with normal serum. This conditioned serum was used in different neutrophils for priming, and these cells were analyzed for PR3 membrane expression by FACS. Data are means ± SEM (given as ratio to buffer-incubated normal serum [–], which was set at baseline to 100%). **P < 0.01.
C3a- and C5a-Conditioned Serum Primes Neutrophils for ANCA-Induced Respiratory Burst
We next tested whether conditioned serum primes neutrophilsfor ANCA-induced respiratory burst. After neutrophil incubationwith conditioned serum for 45 min and subsequent stimulationby anti-PR3 IgG for an additional 45 min, we measured reactiveoxygen species (ROS) production by the dihydrorhodamine (DHR)assay. Figure 3 indicates that normal serum co-incubated withsupernatants from ANCA-stimulated cells was sufficient to primefor ANCA-mediated respiratory burst activity. In contrast, serumtreated with supernatants from control IgG-stimulated polymorphonuclearleucocytes did not result in priming. Similar results were obtainedwhen the ferricytochrome C assay was used. Co-incubation ofnormal serum with supernatants of TNF-–primed neutrophilsactivated with a mAb to MPO yielded 171 ± 37%, comparedwith 124 ± 31% after stimulation with matching isotypecontrol, given as ratio to medium-conditioned serum. These dataindicate that C3a- and C5a-conditioned serum primes human neutrophilsfor subsequent ANCA-induced respiratory burst.
Figure 3. Normal TNF-–primed neutrophils were stimulated as described already. Supernatants from these cells were incubated for 15 min with normal serum. This conditioned serum was used in different neutrophils for priming (45 min at 37°C), after which cells were activated with a mAb to PR3. ROS production was measured after 45 min of stimulation with PR3, and data are means ± SEM (given as ratio to buffer-incubated normal serum [–], which was set at baseline to 100%). **P < 0.01.
Priming with Activated Serum Requires Complement Factor C3 and C5 and Is Mediated by the C5aR on Neutrophils
We next wanted to establish firmly that the observed primingis indeed mediated by the complement system and not by otherfactors generated in the serum treated with supernatants fromANCA-stimulated neutrophils. Furthermore, we explored whichof the two complement components exercises direct effects onneutrophils via its specific receptor. We first confirmed thatneutrophils express both C3aR and C5aR by flow cytometry. Atypical flow cytometry analysis is depicted in Figure 4, A andB. In addition to normal serum, we next used C3- and C5-depletedserum to prepare conditioned sera. Figure 4C demonstrates thatthe presence of either C3 or C5 was essential to generate conditionedserum capable of neutrophil priming for an ANCA-induced respiratoryburst. When C3- or C5-depleted serum was used instead of normalserum, the priming capability for subsequent activation of respiratoryburst activity in response to a mAb to PR3 was completely abrogated.The priming capability was restored after C5-depleted serumwas repleted with human C5 (data not shown); however, C3 isupstream of C5. Conceivably, C3 is needed only for C5a generationas opposed to a direct role in ANCA-induced activation priming.Thus, we used specific receptor blockade for either C3aR orC5aR, respectively, and observed complete abrogation of ANCA-inducedrespiratory burst activity when a C5aR blocker was present duringpriming with conditioned serum, as shown in Figure 4D. We confirmedthe effect and the specificity of the C5aR-blocking antibody(clone S5/1) by showing complete abrogation of C5a-induced primingfor ANCA-induced H2O2 production without inhibition when TNF-was used for priming. Using the DHR assay and flow cytometry,control IgG-treated cells resulted in 15 ± 0.4 mean fluorescenceintensity (MFI) after TNF- priming and in 12.0 ± 0.4MFI after C5a priming. These values increased with PR3-ANCAto 180.0 ± 39.0 with TNF- priming and to 122.0 ±38.0 with C5a priming. Preincubation with the C5aR-blockingantibody had no effect in PR3-ANCA–stimulated neutrophilsprimed with TNF- (167.0 ± 45.0 MFI), whereas the MFIwas almost completely abrogated to 23.0 ± 5.0 with C5apriming (n = 3). In contrast, the specific C3aR antagonist SB290157showed no effect. These data suggest that C3 is necessary forC5a generation; however, it is the C5a that primes neutrophilsvia the C5aR.
Figure 4. (A and B) Normal neutrophils were tested for C3aR and C5aR expression by FACS. A representative example is shown. The gray line depicts the matching isotype control. (C) TNF-–primed neutrophils were stimulated with a mAb to PR3 for 45 min. Supernatants of these were incubated for 15 min with normal serum (NS), C3-depleted serum, or C5-depleted serum. These different conditioned sera were used in different neutrophils for priming (45 min at 37°C), after which cells were activated with a mAb to PR3. ROS production was measured after 45 min of stimulation with PR3, and data are means ± SEM (given as MFI; five independent experiments performed in duplicate). (D) Neutrophils were preincubated with a blocking antibody to C5aR or a C3aR blocker, afterward primed with conditioned serum for 45 min and subsequently activated with a mAb to PR3. ROS production was measured after 45 min of stimulation with PR3, and data are means ± SEM (given as MFI; data from three independent experiments performed in duplicate). **P < 0.01.
Reactive Oxygen Radicals Are Mediators for Complement Activation
We next explored mechanisms. We reasoned that a protease- ora ROS-mediated mechanism could be responsible. Thus, we addedPMSF or superoxide dismutase (SOD) during the neutrophil stimulation.Furthermore, we heat-inactivated the supernatants for 10 minat 56°C before mixing with serum. As shown in Figure 5,SOD significantly prevented the priming effect of the supernatant-conditionedserum, whereas PMSF or heat inactivation did not. Thus, ourdata support the notion that ROS are important mediators ofcomplement activation.
Figure 5. Normal TNF-–primed neutrophils were stimulated as described already. Supernatants from these cells were incubated with normal serum. Before co-incubation, PMSF or SOD was added to the supernatants, or supernatants were heat inactivated. This conditioned serum was used in different neutrophils for priming, before analysis of cells for membrane-PR3 expression by FACS. Data are means ± SEM (given as ratio to buffer-incubated normal serum [–], which was set at baseline to 100%). **P < 0.05.
Recombinant C5a Primes Neutrophils Dosage Dependently for ANCA-Induced Respiratory Burst
We next studied whether C5a is a sufficient primer for ANCA-inducedneutrophil activation. Additional yet not identified serum factorscould be needed for the C5a effects. We used recombinant C5ato prime neutrophils for 15 min with increasing concentrationsfollowed by subsequent activation with a mAb to MPO. We quantifiedsuperoxide production using the continuous ferricytochrome Cassay. Figure 6A shows a dosage-dependent C5a priming effectranging from 10–7 to 10–9 M. We observed no burstactivity with C5a alone, even at the highest concentration.The effect of priming with TNF- was performed in parallel andis shown for comparison. In the next set of experiments, weused human ANCA preparations instead of mAb to MPO. As shownin Figure 6B, recombinant C5a (5 x 10–8 M) primed neutrophilsfor respiratory burst activity in response to MPO- and PR3-ANCAIgG preparation, but not for control IgG. Again, TNF- primingis shown for comparison.
Figure 6. (A) Normal neutrophils were primed with buffer (cells), 2 ng/ml TNF-, or C5a in different concentrations for 25 min at 37°C. Afterward, neutrophils were stimulated with an activating mAb to MPO (MPO Ab) or a matching isotype control (isotype mAb). ROS production was measured by the ferricytochrome assay, and data are the 45-min time point (data from four independent experiments performed in duplicate). (B) Neutrophils were primed as described already and subsequently activated with 125 µg/ml normal IgG (Ctrl IgG), PR3-ANCA IgG (PR3 IgG), or MPO-ANCA IgG (MPO IgG); activation was measured by the ferricytochrome assay, and data are the 45-min time point. , Unprimed cells; , C5a-primed cells; , TNF-–primed cells (data from three independent experiments each using two different PR3-ANCA IgG, two different MPO-ANCA IgG, and two different normal IgG, done in duplicate).
Taken together, these in vitro experiments demonstrate thatneutrophils release factors after stimulation with ANCA thatactivate the complement cascade in serum, resulting in the generationof C3a and C5a. This conditioned serum primes neutrophils forsubsequent ANCA-induced activation. The observed direct primingeffects on neutrophils are caused by C5a, rather than C3a, andare mediated via the neutrophil C5aR.
C5aR-Deficient Animals Are Protected from ANCA-Induced NCGN
We next studied the role of C5a in ANCA-induced NCGN in an invivo model. Anti-MPO induced NCGN was induced by the previouslydescribed BM transplantation approach.9 MPO-deficient mice wereimmunized with MPO and irradiated and received BM cells fromWT mice or C5aR-gene deficient (C5aR–/–) mice (n= 6 in the WT group, n = 8 in the C5aR–/– group).Eight weeks after transplantation, mice were killed. All mice(100%) that received WT BM cells demonstrated signs of renaldisease by developing hematuria and proteinuria. In contrast,mice that received C5aR–/– BM did not develop significanturine abnormalities. These results were confirmed by a significanthigher urinary albumin excretion in the mice that received WTBM in comparison with the C5aR–/– BM group: 54.0± 13.4 µg/ml in the WT mice compared with 10.0± 1.5 µg/ml in the C5aR–/– mice (P< 0.05).
All mice that received WT BM developed NCGN on histology (100%disease induction), whereas mice in the C5aR–/–BM group developed only very weak glomerular abnormalities,as shown in Figure 7, A through E. with further details in Table 1.Immunohistology demonstrated a weak accumulation of IgG, IgA,and IgM and complement C3 that did not differ from untreatedmice (data not shown). A potential influence of the C5aR phenotypeon the anti-MPO IgG level was ruled out because we observedsimilar titers in both groups as assessed by ELISA (anti-MPOtiter after BM transplantation: day 0 WT 2.0 ± 0.6 versusC5aR–/– 2.0 ± 0.2; day 28 WT 1.8 ±0.6 versus C5aR–/– 1.5 ± 0.4; day 56 WT 1.2± 0.8 versus C5aR–/– 1.3 ± 0.3). Inaddition, both groups of mice demonstrated a similar engraftmentwith MPO-positive cells.
Figure 7. Mice were killed 8 wk after BM transplantation, and renal tissue was examined. (A) The extent of glomerular crescents and necrosis was expressed as the mean percentage of glomeruli with crescents and necrosis. (B) Glomerulus from a C5aR–/– mouse with no lesion.(C through E) WT mice developed glomerular crescents and necrosis. (F and G) Examination for glomerular neutrophil infiltration showed that C5aR–/– mice had fewer glomerular neutrophils (F) than did WT mice (G). **P < 0.01.
Table 1. Pathologic findings in MPO-deficient micea
Glomerular Neutrophil Influx in Anti-MPO–Induced NCGN Was Diminished in C5aR–/– Mice
We observed glomerular neutrophil accumulation in anti-MPO NCGNmice that received WT BM. Because C5aR mediate neutrophil recruitment,we compared neutrophil glomerular accumulation. This was partlyabrogated in mice that received C5aR–/– BM, as shownin Figure 7 and detailed in Table 2, indicating that the C5aRexpressed on circulating neutrophils plays an important rolefor the glomerular neutrophil accumulation and for the inductionof NCGN.
All three complement cascades lead to C3 convertases that generateC3b and C3a by C3 cleavage. Further downstream, C5b and theC5b-9 MAC that mediates complement-induced effects are formed,which are involved in the pathogenesis of renal diseases, suchas membranous nephropathy, mesangioproliferative glomerulonephritis,and membranoproliferative glomerulonephritis.12–15 Activationof the complement cascade also yields the small cleavage productsC3a and C5a. These anaphylatoxins function as potent inflammatorymediators via specific receptors on both myeloid and nonmyeloidcells. We showed that the generation of the anaphylatoxin C5aby supernatants from ANCA-stimulated neutrophils and C5a-mediatedpriming effects via its specific receptor allowed neutrophilsto generate ROS in response to ANCA. These data suggest a previouslyunknown role for C5a in ANCA-induced neutrophil activation.We then extended our studies to an animal model of ANCA-inducedNCGN. Lack of C5aR on BM-derived circulating cells significantlyinhibited neutrophil recruitment and almost completely preventedglomerulonephritis in this neutrophil-dependent mouse modelof ANCA-induced disease. Our data outline an amplification loopthat may provide a novel therapeutic target in ANCA-associatedNCGN.
A role for complement in the induction of ANCA-mediated glomerulonephritiswas demonstrated in two recent animal studies. Xiao et al.10proved that the alternative pathways of complement activationare essential for disease induction, whereas Huugen et al.11showed that anti-MPO NCGN could be prevented by inhibition ofcomplement factor C5. ANCA NCGN in humans is characterized bya paucity of Ig and complement deposits; however, many patientshave demonstrable accumulations of complement factors and Igin glomeruli.16 From the clinical and pathologic characteristicsof patients with ANCA vasculitis and NCGN, we reasoned thatcomplement effects mediated by MAC deposition within the glomeruliwere less likely to play a significant role in the disease process.We considered an alternative role for complement and studiedgeneration and biologic effects of anaphylatoxins in ANCA-NCGNinduction. Anaphylatoxins are involved in a variety of highlyinflammatory processes, and neutrophils along with other myeloidcells express C3aR and C5aR.
We first learned from a series of in vitro experiments thatANCA-stimulated neutrophils release substances that activatethe complement cascade in the serum. In addition to C3a generation,a phenomenon that we had observed in a previous study,10,11we found C5a generation to be present as well. Sonicated neutrophilsor stimulated intact neutrophils were found to activate thecomplement system in human plasma with generation of C5a andC5b-9.17 Other investigators identified specifically MPO andsuperoxide as complement activators.18–20 In addition,neutrophils harbor proteases that are also capable of activatingthe complement system.21,22 Conceivably, these candidates arealso at work in ANCA-stimulated neutrophils. We identified ROSreleased by neutrophils as a source of activator of the complementsystem. Complement activation was significantly inhibited bytreatment with SOD; however, because we observed only partialinhibition, additional factors released are presumably at work.Properdin could be a participant because activated neutrophilsrelease biologic active properdin.23
We used sera depleted of C3 or C5, recombinant C3a and C5a,specific C3aR and C5aR blockers to establish the importancefor both anaphylatoxins in ANCA-induced neutrophil activationin vitro. Our data indicate that C5a but not C3a upregulatesthe membrane expression of ANCA antigens and primes neutrophilsfor a subsequent ANCA-induced respiratory burst. A role forC5aR has been demonstrated in other glomerular diseases.24 C5ais one of the most potent inflammatory peptides with a broadspectrum of actions implicated in many types of inflammation.The molecule shows activities toward many cell types, but themain action seems to be on myeloid cells. For example, C5a isa potent chemoattractant for neutrophils, monocytes, and macrophages.25In addition, C5a prolongs neutrophil survival by delaying apoptosis,26enhances expression of adhesion molecules on neutrophils,27activates respiratory burst and phagocytosis,28 and participatesin degranulation.29 All of these functions are important forinflammatory responses and have implications in terms of ANCA-mediatedtissue damage. Conceivably, in addition to the upregulationof ANCA antigens and priming for the ANCA-induced respiratoryburst shown in our in vitro experiments, some or all of theseC5a-related functions participate in ANCA-mediated neutrophilinflammation in vivo.
C5a exerts its action through two different receptors: C5aR(CD88) and C5L2 (GPR77).30,31 Both receptors feature seven transmembranedomains and bind C5a with high affinity. Most of the C5a actionsare mediated by the C5aR, and only a few biologic responsesare mediated by the more modestly expressed C5L2.31,32 The C5L2receptor might serve as a decoy receptor for C5a without anybiologic effect; however, an anti-inflammatory function33 andrecently a proinflammatory action have been suggested.34,35
We had an excellent mouse model of ANCA-mediated NCGN availableto supplement our in vitro data and tested the significanceof the C5aR in this model. This model is particularly suitablefor studying neutrophil-dependent functions during disease induction.Previous studies showed that neutrophil depletion completelyprevented NCGN in this model.36 In our experiments, we reconstitutedthe irradiated mice with either WT or C5aR–/– BMfrom WT. This approach allowed us to study whether the C5aRon myeloid cells was important. Our data demonstrate that thelack of C5aR on myeloid cells significantly reduced urinaryabnormalities, neutrophil recruitment, and renal damage.
Collectively, our study demonstrates that C5aR expressed onneutrophils is essential in the pathogenesis of ANCA-inducedNCGN. We propose that complement activation is initiated byANCA-induced neutrophil activation and leads to the generationof C5a, which attracts and primes further neutrophils for fullactivation in response to ANCA. The engagement of the C5a–C5aRpathway is an extremely important event in the pathogenesisof ANCA-induced NCGN. A pharmacologic intervention might providea novel and less toxic therapeutic strategy. We are currentlypursuing these ideas.
TNF- was obtained from R&D Systems (Wiesbaden-Nordenstedt,Germany). Phorbol-2-myristate-13-acetate, DHR, and Ficoll-Hypaquewere from Sigma (Deisenhofen, Germany); recombinant C5a wasfrom Biovision (Mountain View, CA); and recombinant C3a andSB290157 were from Calbiochem (Darmstadt, Germany). The blockingantibody to CD88 (C5aR) was clone S5/1 from AbD Serotec (Düsseldorf,Germany).37 Complement-depleted sera were from Merck Biosciences(Darmstadt, Germany). HBSS, PBS, and trypan blue were from Biochrom(Berlin, Germany), and dextran was purchased from GE Healthcare(Amsterdam, Netherlands). The PR3 mAb 4A5 was from Wieslab (Lund,Sweden), and the mAb to MPO was from Acris (Hiddenhausen, Germany);horseradish peroxidase–labeled donkey anti-rabbit IgGwas from GE Healthcare. Endotoxin-free reagents and plasticdisposables were used in all experiments.
Preparation of Human Neutrophils and Human IgG
Neutrophils from healthy human donors were isolated from heparinizedwhole blood as described previously.38 Cell viability was detectedin every cell preparation by trypan blue exclusion and foundto be >99%. Wright-Giemsa staining revealed a neutrophilpercentage after isolation >95%. Normal IgG and ANCA-IgGwere prepared from normal volunteers and patients with MPO andPR3-ANCA with active disease using a High-Trap–proteinG column in an Äkta-FPLC system (both from GE Healthcare).A single band was detected on Coomassie-stained gels.6
Measurement of Respiratory Burst by DHR Oxidation to Rhodamine
We assessed the generation of reactive oxygen radicals usingDHR as described previously.39 In brief, neutrophils (1 x 107/mlHBSS) were loaded with DHR (1 µM) for 10 min at 37°C.After 40 min of priming with 2 ng/ml TNF- or conditioned serumas described in the section titled Activation of Human Neutrophilsand Incubation of Serum with Their Supplements, cells were dividedand 5 x 105 cells were incubated with the stimuli in a totalassay volume of 100 µl. Indicated antibodies were added,and the reactions were stopped after another 30 min by additionof 400 µl of ice-cold PBS/1% BSA. We analyzed samplesusing a FACScan (Becton Dickinson, Heidelberg, Germany). Datawere collected from 10,000 cells per sample. The shift of greenfluorescence in the FL-1 mode was determined. For each condition,the MFI (representing the amount of generated hydrogen peroxide)is reported.
Measurement of Respiratory Burst by Reduction of Ferricytochrome C
We measured superoxide using the assay of SOD-inhibitable reductionof ferricytochrome C as described by Pick and Mizel.40 We pretreatedneutrophils with 5 µg/ml cytochalasin B for 15 min. Cells(0.75 x 106) were primed with 2 ng/ml TNF- or conditioned serumfor 45 min before human ANCA preparations or mAb to MPO or PR3were added. The final concentrations were 125 µg/ml forpurified IgG preparations and 10 µg/ml for mAb. We conductedexperiments in duplicate. Samples were incubated in 96-wellplates at 37°C for up to 60 min, and the absorption of sampleswith and without 300 U/ml SOD was scanned repetitively at 550nm using a Microplate Autoreader (Molecular Devices, Munich,Germany). The final ferricytochrome C concentration was 50 µmol/L,and the final cell concentration was 3.75 x 106/ml.
Surface Antigen Expression by Flow Cytometry
We used FACS as described previously to evaluate the surfaceexpression on neutrophils.6 The following antibodies were used:MAb to PR3 (CLB 12.8, Amsterdam, Netherlands), MPO (Acris, Hiddenhausen,Germany), CD88 (C5aR, AbD Serotec, Düsseldorf, Germany),C3aR (AbD Serotec, Düsseldorf, Germany), and an appropriatematching isotype control. Flow cytometry was performed usinga FACScan (Becton Dickinson, Heidelberg, Germany), and 10,000events per sample were collected.
Activation of Human Neutrophils and Incubation of Serum with Their Supernatants
Neutrophils were treated for 15 min at 4°C with cytochalasinB, primed for 15 min at 37°C with TNF-, and subsequentlyactivated with human ANCA IgG or control IgG for an additional15 min. Cells were centrifuged for 7 min at 300 x g, and thesupernatants were centrifuged again for 7 min 1000 x g. In supernatantsthat were not previously stimulated with ANCA IgG, ANCA IgGwas added to achieve the same concentration in all samples.Supernatants and normal serum (AB-serum; Sigma-Aldrich) wereco-incubated for 45 min at 37°C 1:1 (50 µl/50 µl).In the inhibition studies, 0.5 mM PMSF or 300 U/ml SOD was addedduring the neutrophil stimulation. For heat inactivation, supernatantswere heated for 10 min at 56°C before co-incubation withserum. For the repletion studies, C5-deficient serum was repletedwith human C5 (Calbiochem 234405) to a final concentration of75 µg/ml and with CaCl2 to a final concentration of 20mmol/L. Nafamostat mesilate (Futhan) was added afterward toprevent further complement activation, and samples were eitherfrozen at –80°C until further testing or instantlyused for further experiments.
C5a ELISA
C5a generation was measured by a C5a-specific ELISA (BD Bioscience,Heidelberg, Germany). In brief, normal serum was co-incubatedwith supernatants from neutrophils activated with human ANCAIgG or control IgG as described already. This conditioned serumwas tested for generation of C5a in a C5a-specific ELISA. C5alevels were calculated with a standard curve of different C5aconcentrations.
Mice
C57BL/6J (B6) mouse breeding pairs were purchased from JacksonLaboratories (Bar Harbor, ME). Mice lacking MPO (MPO–/–mice) were the sixth-generation progeny of a backcross intoB6 mice originally generated by Aratani et al.41 MPO–/–mice (8 to 10 wk old) were used for immunization. Nine- to 10-wk-oldB6 WT mice and C5aR–/– mice (gift from Prof J.E.Gessner, Department of Clinical Immunology, Medical School Hannover,Hannover, Germany) were used as BM donors. Local authoritiesapproved all animal experiments, which followed American PhysiologicSociety guidelines for animal care.
Immunization of Mice
The purification of mouse MPO and the immunization of MPO–/–mice were performed as described previously.7 Briefly, mouseMPO was purified from WEHI-3 cells by Dounce homogenization,Concanavalin A affinity chromatography, ion exchange, and gelfiltration chromatography. MPO–/– mice were immunizedintraperitoneally with 10 µg of purified murine MPO incomplete Freund's adjuvant. Development of antibodies was monitoredby anti-MPO ELISA. The presence of circulating anti-MPO antibodieswas confirmed in selected mice by indirect immunofluorescencemicroscopy assay on murine neutrophils as described previously.7
BM Transplantation in Mice
After immunization, the MPO–/– mice were kept insterile housing conditions with food and water ad libitum (sterilewater with trimethoprim and sulfadoxine; Borgal, Intervet, Unterschleissheim,Germany). After immunization, mice were -irradiated with 900rad of whole-body dose and reconstituted with BM cells fromWT or C5aR–/– mice of the same genetic background.BM cells were harvested from femurs and tibia, erythrocyteswere lysed, and 1.5 x 107 BM cells were intravenously injected.Engraftment was measured by histochemistry for MPO activityon peripheral blood smears.
Functional Evaluation of Renal Injury
Mice were placed in metabolic cages 1 d before being killed,and urine was collected for 12 h overnight. Urine was testedby dipstick (Roche Diagnostics, Indianapolis, IN) for hematuria,leukocyturia, and proteinuria, and the extent is expressed asthe mean on a scale of 0 (none) to 4 (severe). The albuminuriawas determined by an albumin-specific ELISA (CellTrend, Lukenwalde,Germany).
Histologic Evaluation of Renal Injury
Samples of kidney tissue were collected when mice were killed,fixed in 10% formalin, and embedded in paraffin using routineprotocols. Four-micrometer coronal sections of specimens werestained with hematoxylin and eosin and periodic acid-Schiffand evaluated by light microscopy. The extent of glomerularcrescents and necrosis was expressed as the mean percentageof glomeruli with crescents and necrosis in each mouse. Forimmunofluorescence microscopy to detect glomerular localizationof immune determinants, 4-µm frozen sections were stainedwith fluoresceinated antibodies. The glomerular IgG depositionwas performed by staining using FITC-conjugated goat anti-mouseIgG (Molecular Probes Invitrogen, Carlsbad, CA). Depositionof mouse complement C3, IgG, IgM, IgA, and MPO was visualizedwith a FITC-conjugated goat anti-mouse C3, IgG, IgM, IgA, andMPO (ICN/Cappel, Aurora, OH). Immunofluorescence microscopystaining of glomeruli was expressed as the mean intensity ofstaining for IgG, IgM, IgA, C3, and MPO on a scale of 0 to 4+.Glomerular neutrophil influx was visualized using a neutrophil-specificmAb to GR-1 (BD Bioscience, Heidelberg, Germany) as describedpreviously.9
Statistical Analysis
Results are given as means ± SEM. Comparisons betweentwo groups were done using paired t test. Comparisons betweenmultiple groups were done using one- or two-way ANOVA as indicated.Specific differences between multiple groups were then determinedby use of a Bonferroni post hoc test. Differences were consideredsignificant at P < 0.05.
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Top
Abstract
Introduction
and subsequently stimulated them with human MPO-ANCA or PR3-ANCA IgG, resulting in neutrophil activation. We documented activation by estimating the degranulation response, measured as β-glucoronidase release and respiratory burst, measured as superoxide release. Degranulation was 5.7 ± 0.7% in unstimulated cells, 4.9 ± 2.0% in control IgG-treated cells, 15.0 ± 5.4% after treatment with MPO-ANCA, and 27.1 ± 13.0% (n = 4; P < 0.05) with PR3-ANCA. Superoxide production was 2.0 ± 0.6 nmol/106 neutrophils in unstimulated cells, 4.0 ± 0.6 in control IgG-treated cells, 19.0 ± 2.5 after treatment with MPO-ANCA, and 20 ± 3.0 after treatment with PR3-ANCA (n = 4; P < 0.05). We then co-incubated the supernatants for 15 min with an equal volume of normal serum (from here on called conditioned serum). Figure 1 shows C5a generation in the conditioned sera as measured by ELISA. These data show that activation of TNF-
-irradiated with 900 rad of whole-body dose and reconstituted with BM cells from WT or C5aR–/– mice of the same genetic background. BM cells were harvested from femurs and tibia, erythrocytes were lysed, and 1.5 x 107 BM cells were intravenously injected. Engraftment was measured by histochemistry for MPO activity on peripheral blood smears. 
, Unprimed cells; 
, C5a-primed cells; 
, TNF-