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The handle http://hdl.handle.net/1887/48207 holds various files of this Leiden University dissertation
Author: Kotimaa, Juha
Title: Analysis of systemic complement in experimental renal injury and disease Issue Date: 2017-04-25
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Chapter 5
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Properdin binding independent of complement ac- tivation in an in-vivo model of anti-GBM disease.
Juha Kotimaaɸ, Joseph O`Flynnɸ, Ria Faber-Krol, Karin Koekkoek, Ngaisah Klar- Mohamad, Angela Koudijs, Wilhelm J. Schwaeble, Cordula Stover,
Mohamed R. Daha, Cees van Kooten ɸ Authors have contributed equally.
In preparation
ABSTRACT
Anti-glomerular basement membrane (anti-GBM) disease is a rare autoimmune disease resulting in glomerular injury and loss of renal function. Clinical and animal studies have established that neutrophils, classical (CP) and alternative (AP) pathways of the complement system contribute to the pathogenesis of the disease. Here, we studied the role of properdin, the positive regulator of AP, in an experimental mouse model of anti-GBM disease. At 2 hours following administration of anti-GBM, mice showed significant increase of C3 activation fragments in circulation, accompanied by a consumption of all three complement pathways. This consumption was not observed in properdin-KO mice and these mice showed no detectable AP activity. Despite reduced complement activation, in this model properdin-KO, but also C3-KO mice, were not protected against renal injury, as assessed by urinary protein excretion. Nevertheless, kinetic analysis showed that both C3 and properdin deposition were already observed early upon injection (2 hours) and increased over time (until 72 hours), whereas deposition of C9 was a late event, and was prominent from 48 hr onwards. Neutrophil infiltration and activation was an early and transient process, but was similarly present in properdin-KO mice.
Histological analysis revealed that properdin was present in the glomeruli already by 2 hours, and showed only partial colocalisation with neutrophils, C3 or disease inducing IgG. Importantly, glomerular properdin deposition was also present in C3 KO mice. Together these results demonstrate for the first time that, also in vivo, properdin can bind to injured tissues independent of C3 deposition, supporting the model of properdin-directed AP activation.
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1. INTRODUCTION
Anti-glomerular basement membrane (anti-GBM) antibody disease is a rare glomerular vasculitis, known earlier as Goodpasture’s syndrome or disease.
Anti-GBM disease is characterised by autoantibody deposition on the glomerular basement membrane, initiation of complement and neutrophil activation resulting in rapid loss of renal function [1, 2].
The complement system consists of three activation pathways, classical (CP), lectin (LP) and alternative (AP) which all converge at the level of C3 activation, proceeding to C5 cleavage and initiating the terminal pathway (TP) C5 – C9 activation. The CP is initiated by C1q-C1s-C1r complex binding to ligands such as antibodies. The LP is initiated by binding of mannan binding lectin or ficolins on different sugar moieties, whereas AP greatly amplifies the CP and LP mediated activation and can activate spontaneously on surfaces without sufficient regulators of complement activation [3–6]. Complement activation results in opsonisation, and subsequent phagocytosis of injured cells and pathogens, generation of anaphylatoxins like C3a and C5a, which promote chemotaxis of inflammatory cells to the injury site, and assembly of the C5b-9 lytic complex which promotes apoptosis of injured host cells and lytic killing of pathogens [5, 6].
The role of properdin, a component of AP and the only positive regulator of the complement system, has remained enigmatic ever since its discovery and characterisation [7–9]. It has been proposed that properdin might have a dual role both as a positive regulator as well as a recognition molecule. However, it is not yet understood how properdin could fulfil its postulated recognition function, since it lacks the specific recognition domains which have been identified to be responsible for ligand binding in other complement factors such as C1q, MBL, CL-11 and ficolins [10–13]. Still, in vitro studies have established
oxidized-LDL and myeloperoxidase [14–16]. Moreover, a similar mechanism has been demonstrated on apoptotic and necrotic cells, and heparan sulfates expressed at the surface of renal epithelial cells [17–20]. It has been hypothesised that this mode of AP activation involves initial binding of properdin to a ligand, followed by the subsequent binding of C3b, factor B and factor D resulting in formation of a properdin-initiated AP C3 convertase [9, 14, 17].
Recent studies in anti-GBM disease have described that properdin, and other AP components are prominently present in anti-GBM affected glomeruli in humans [21, 22]. However, in the context of anti-GBM disease, it is not known whether properdin directly activates the AP by binding directly to injured glomeruli or whether properdin stabilises and prolongs the half-life of AP C3 convertase C3bBb [23].
To date, the contribution of the AP and properdin in tissue injury and disease pathologies has been studied either with therapeutic intervention or with recently developed genetic knockout mice, establishing a role for properdin in renal I/RI, abdominal aortic aneurysm and arthritis [24–27]. Previous studies with different experimental anti-GBM models have shown that, in addition to Fc-mediated activation of neutrophils and CP activation, also the AP contributes to the renal injury, which is in line with clinical observations [22, 28, 29].
To better understand the role of properdin in anti-GBM disease, we used a mouse model of anti-GBM and evaluated complement activation and renal injury in wild type, C3 KO and properdin KO mice, and applied novel methods to investigate mouse complement in tissues and in serum [30]. Our present results demonstrate that properdin binds injured glomeruli independently of C3, which to our knowledge is the first direct evidence of properdin binding to injured host tissue.
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2. MATERIALS AND METHODS
animals used in this study
C57BL/6 wild type (WT) mice were purchased from Charles River laboratories.
C3 knockout (KO) mice were a kind gift from Mike Carroll (Harvard Medical School, Boston, MA, USA), properdin KO mice were generated by Cordula Stover (University of Leicester, Leicester, UK) [31]. All experiments were approved by the Leiden University animal ethical committee and performed according to institutional and national guidelines.
induction of glomerulonephritis
Rabbit anti-rat GBM, cross reacting with mouse GBM, was an in house preparation and cross reactivity was confirmed in pilot experiments with anti- GBM and control antibodies injected into wild-type (WT) C57BL/6 mice.
For the model, both male and female age-matched (ranging from 7-10 weeks) mice (C57BL/6 WT, C3KO mice and properdin KO mice) were used. At day 0, the mice were injected intravenously with rabbit anti-GBM or control rabbit IgG (0.5mg in 200µl physiological saline).
The acute response to anti-GBM administration was evaluated at 2h after injection. WT mice were injected with anti-GBM or control antibody, whereas properdin KO mice were injected only with anti-GBM. For complement determinations, EDTA-plasma was collected by tail-cut from each mouse before the injection and at 2h after injection without anaesthesia. Serum was collected after CO2-anaesthesia via heart puncture. All blood samples were place directly on ice and prepared as plasma or serum as described previously (Kotimaa et al., 2015).
The impact of C3 and properdin deficiency was investigated by injecting either anti-GBM or control IgG (0.5 mg) in WT, C3 KO and properdin KO mice. The severity of renal injury was evaluated by 24h urine collection, and proteinuria analysis from WT (n=10), properdin KO (n=8) and C3 KO (n=9) mice injected with anti-GBM antibody, and WT mice injected with control antibody. For urine collection mice were placed in metabolic cages 72 h after anti GBM administration with access to food and water. The collected urine was centrifuged to remove faeces and other debris, aliquoted and stored at -20°C for further analysis.
To investigate the kinetics of systemic inflammation and complement deposition at the renal level, groups of mice were sacrificed at 2h, 6h, 24h, 48h, and 72h after injection of anti-GBM. The mice were sacrificed by CO2 anaesthesia, kidneys were harvested and snap frozen for later histological analysis. Furthermore, additional kidneys were collected at 2h from properdin KO mice, and at 72h from C3 KO mice that were injected with either anti-GBM or control IgG.
measurement creatinine and proteinuria
Albumin in the urine was quantified using rocket immune-electrophoresis as previously described [28]. Urine creatinine was quantified using creatinine strips for the Reflotron Plus system (Roche Diagnostics, Mannheim, Germany).
For each mouse, the proteinuria was calculated as urinary albumin-creatinine ratio (U-ACR).
properdin ELISA
A specific ELISA for mouse properdin was developed with mouse anti- mouse properdin monoclonal antibody (mAb) (clone 17-17), and rabbit polyclonal antibody (pAb) raised against recombinant mouse properdin [32].
The capture mAb was coated at 2 µg/ml in 96 well ELISA plates (Nunc
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Maxisorp; Thermo Fisher Scientific, Massachusetts, United States). Following coating, the wells were blocked with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). Normal mouse CD-1 serum, recombinant mouse properdin or properdin-deficient serum was serially diluted in PBS, 0.05% Tween 20, 1% BSA (PTB) and added to the blocked wells. Captured properdin was detected using DIG-labelled rabbit anti-human properdin (in house, LUMC), anti-DIG-POD (Prod.no. 11207733910, Roche Diagnostics), followed by colorimetric quantification with a colorimetric substrate step with 3,3’,5,5’-tetramethylbenzidine (TMB) for 15-30 min at room temperature and stopped with 50 µl 1M H2SO4 and read at 450 nm with a BioRad 550 instrument (BioRad, Tokyo, Japan).
complement activation fragments and functional complement analysis
Measurement of functional mouse complement pathway activities was performed with an ELISA based system as described earlier [30, 33]. In brief, human IgM coated plates were used for CP ELISA, Mannan coated for LP, and LPS coated plates for AP. For CP and LP assessment plasma samples were diluted in MgCl2 and CaCl2 supplemented Veronal buffered saline, and for AP samples Veronal buffer supplemented with MgCl2 and C-chelating EGTA.
As a measure of complement activity up to C3, deposition of activated mouse C3 functional ELISAs was detected with biotinylated rat anti-mouse C3 mAb clone 2/11, which is specific for C3 activation fragments C3b, C3c and iC3b (Mastellos et al., 2004, HM1065, Hycult Biotech, Uden, the Netherlands) and Streptavidin-HRP conjugate (Hycult Biotech).
determination of activated mouse C3, MPO and SAP
Commercial assays were used to measure SAP (HK215, Hycult Biotech) and MPO (HK210, Hycult Biotech) according to manufacturer’s specifications.
The C3b/C3c/iC3b ELISA, as a measure of activated C3, was performed as described previously [30]. In short, clone 2/11(HM1065, Hycult Biotech), directed against C3b/iC3b/C3c, was coated at 3 µg/ml on Nunc Maxisorp plates overnight (Thermo Fisher Scientific). After washing samples were incubated on the plate in PBS/EDTA buffer at 4°C for 1h with following steps 1h at 37°C.
Bound C3b/C3c/iC3b fragments were detected with DIG-conjugated Rabbit anti-mouse C3 (In house) and Rabbit anti-DIG-POD (Roche Diagnostics).
Zymosan activated mouse serum was used as a standard for quantification of C3b/C3c/iC3b in experimental samples.
histological analysis
Kidney sections were sectioned into 4 µm slices using a cryostat and fixed by 10 min incubation in acetone, washed with PBS three times 5 min after each step and all antibodies were diluted in 1%BSA/PBS.
Mouse properdin and mouse C9 were detected using tyramide-fluorescein isothiocyanate (FITC) amplification based staining. The slides were first treated with 45 min RT incubation in PBS buffer containing 0.6% H2O2 (1.07209.0250, Merck KGaA, Darmstad, Germany) and 0.2% NaN3 (1.06688.0100, Merck) diluted in PBS.
Properdin was detected with 1µg/ml diluted rabbit anti-mouse properdin-DIG described for the properdin ELISA (in house, LUMC), whereas mouse C9 was detected with 1µg/ml rabbit anti-mouse recombinant C9-DIG described previously [30]. For both components, the detection was followed with
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over-night incubation of the slides with 1/750 diluted sheep anti-DIG-POD (Roche Diagnostics). Finally, the slides were incubated 20 min with 1/500 diluted tyramide-FITC (T9034-4, Sigma-Aldrich, Missouri, United States) in tyramide buffer (NENTM, Life Science Products, Boston, United States) with 0.01% H2O2 (Merck).
Mouse GR1 was detected with 1/400 diluted rat anti-mouse GR1 (a kind gift from professor Georg Kraal, VUMC, Amsterdam, the Netherlands) and 1/750 diluted goat anti-rat-Alexa 568 (A11011, Molecular Probes, Oregon, United States). Deposition of rabbit IgG to mouse glomeruli was detected with 1/400 diluted goat anti-rabbit IgG-Alexa 488 (A11008, Molecular probes, Eugene, United States) or 1/400 diluted goat anti-rabbit IgG-Alexa568 (ab175471, Abcam, Cambridge, United Kingdom). Mouse C3 was detected with 1/1000 rat anti-mouse C3 (CL75603AP, Cedarlane, Burlington, Canada), and 1/750 diluted goat anti-rat Alexa 568 (A11011, Molecular Probes). Where applicable, nuclear stains were performed with 1/10000 diluted Hoechst (H3569, Invitrogen, California, United States) according to manufacturer’s instructions. Fluorescence microscopy was performed with Zeiss Axio Scope A1 with EX-plan Neofluar 40x lens and captured with Axiocam MRC5 (Carl Zeiss LLC, United states).
In all cases specificity of the staining was confirmed with tissues from mice harvested 72h after injection of antibody. Rabbit IgG and C9 stain specificity was controlled with tissues from mice injected with control rabbit IgG. Properdin and C3 staining specificity was evaluated with tissue from properdin-KO and C3-KO mice injected with anti-GBM antibody. In all cases the staining was free of non-specific signal within the glomeruli, as assessed by isotype controls.
3. RESULTS
effect of injection anti-GBM on mouse complement
The purpose of this study was to assess the mechanism and contribution of AP and properdin-mediated activation of complement in the pathogenesis of anti-GBM disease. Direct comparison of mouse plasma, pre- and 2 hours post-administration of anti-GBM, showed significantly increased C3b/C3c/
iC3b in WT mice (3.6 fold), which was not seen following administration of control IgG (Fig 1A). This increase was not observed when anti-GBM was given to properdin-KO mice. Moreover, basal levels of C3 activation fragments was two-fold lower in properdin KO mice as compared to the WT mice.
To be able to monitor properdin levels, we developed a novel mouse properdin ELISA, which showed linear detection range with recombinant mouse properdin (Fig 1B) and no measurable background in plasma of properdin-KO mice (Fig 1C). Analysis of plasma properdin 2h after injection of WT mice either injected with anti-GBM or control antibody did not reveal any changes in circulating levels of properdin (Fig 1C).
Determination of functional complement activities up to C3, before and after anti-GBM antibody injection in WT mice, revealed prominent consumption of CP (Fig 1D) and AP (Fig 1E), whereas for LP consumption was observed in two out of four mice (Fig 1E). This complement consumption was not observed after the injection of control Ab in WT mice. Importantly, in properdin-KO mice administration of anti-GBM did not result in significant CP or LP pathway consumption, and AP activity was already below the detection limit in the pre-sample (Fig 1F).
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Figure 1. Analysis of systemic complement. Evidence of complement activation at 2h after model induction was studied with paired pre – post samples from anti-GBM and control pAb treated WT C57BL/6 mice and anti-GBM treated properdin KO mice, and samples collected at 2h. A) Plasma C3b/C3c/iC3b was determined with sandwich ELISA suggesting acute activation of serum complement after anti-GBM injection. B) Next, properdin sandwich ELISA was developed and its performance was assessed with reciprocal dilutions of recombinant mouse properdin with fourfold steps from 1000 ng/ml followed by C) serum properdin levels determination 2h after administration of anti-GBM in WT and properdin (fP) KO mice, and in WT mice with control antibody injection. To evaluate pathway specific consumption serum D) classical (CP) E) lectin (LP) and F) alternative (AP) pathway activities were determined with functional C3 ELISAs. Two way ANOVA was used to determine change specificity in pre – post analysis, one-way ANOVA was used to determine specificity of change between control and anti-GBM antibodies. Error bars represent standard deviation.
glomerular injury and proteinuria is not prevented in C3- and Properdin KO mice
The results described above indicate that the early stage of anti-GBM disease is associated with complement activation and suggests a role for properdin.
Therefore we evaluated the impact of properdin deficiency on the pathogenesis anti-GBM disease, with urinary albumin-creatinine ratio (U-ACR) as a functional read-out (Fig 2). Injection of anti-GBM in WT C57bl/6 resulted in significant albuminuria in 24h-urine collected between 72h – 96h. Although
variation in disease severity in this model. Whereas properdin-KO and C3-KO mice showed 36 – 43% lower level of albuminuria (U-ACR of 4.3 ± 2.0 and 4.8 ± 2.2 respectively), compared to wild type mice (U-ACR of 6.9 ± 3.7), this was not significantly different. The impact of gender was evaluated, showing that both WT and C3-KO female mice exhibited higher degree of renal injury than male mice, and in general female mice had higher albumin and creatinine concentration in their urine compared to male mice (supplementary table 1).
Figure 2. Anti-GBM induced proteinuria. Anti-GBM disease was induced with injection of 0.5 mg of rabbit anti-GBM IgG in wild type C57BL/6 mice (WT, n=10), properdin-KO mice (fP KO, n=8), and C3-KO mice (n=9).
As a control group, WT C57BL/6 mice were injected with 0.5mg of control rabbit IgG (Ctrl IgG WT, n=5). The gender of each mouse is indicated in the figure as either filled (male) or empty (female) symbols. 24h urine was collected from each mouse in metabolic cages 72-96h for quantification of urinary albumin-creatinine ratio. Non-parametric One-way ANOVA with Dunn’s multiple comparison test was used to determine significance. Error bars represent standard deviation.
Complement activation in the kidneys following anti-GBM administration
To better understand the mechanisms and the kinetics of complement activation in our model, we evaluated the renal deposition following anti- GBM administration in groups of WT mice sacrificed at different time points.
Rabbit IgG was clearly present and persisted in a linear pattern following the GBM at all time points investigated from 2 till 72 hours (Fig 3, IgG). C3
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deposition was present in glomeruli at 2h and increased rapidly. Whereas C3 followed the pattern of rabbit IgG until 48h, at later time points also a focal nonvascular staining pattern emerged (Fig 3, C3). Properdin deposition was already detectable at 2h, however a marked increase was observed from 24h onwards. Interestingly, unlike rabbit IgG and C3, staining for properdin did not show a linear pattern (Fig 3, fP). The staining for C9, as sign of terminal pathway activation, showed a late kinetics of C9 deposition, emerging only after 24h and becoming most prominent at 48h (Fig 3, C9). In all cases specificity of the staining was confirmed and isotype controls showed no staining within the glomeruli. We did not observe gender specific differences in staining pattern or intensity in any of the studied complement factors.
Figure 3. Time course analysis of glomerular activation of complement. Deposition of rabbit anti- GBM IgG was detected with Alexa-488 (green) labelled secondary antibody, showing clear retention of disease inducing antibody in the glomerular vasculature throughout the observation period. C3 deposition was detected with Alexa-568 (red) labelled secondary antibody, showing an initial increase and persistent deposition. Mouse properdin (fP) was detected with HRP-labelled secondary antibody and Tyramide-FITC amplification (green). Stain shows marked increase towards 24h and increased diffusion in the staining pattern. Last, C9 was detected with Tyramide-FITC (green) showing clear increase
neutrophil activation and inflammation after administration of anti GBM
Since complement activation appeared not to be the main driver of albuminuria, we assessed the role of neutrophils, which through the action of Fc-gamma receptors have been shown to contribute to different experimental models of anti-GBM disease[28]. Time course analysis of neutrophils, using GR1 staining, showed that infiltration within the glomeruli was most prominent at 2h, and returned to low levels from 24h to 48h. However, influx of neutrophils appeared to increase again at 72h (Fig 4A). Acute infiltration and activation of neutrophils was supported by a threefold increase in serum MPO at 2-6 h (Fig 4B). The renal injury resulted also in systemic inflammation which was demonstrated by increased serum SAP peaking at 24h and remaining elevated until 72h (Fig 4C). Interestingly, the same time course analysis of serum properdin showed a gradual increase from baseline, which reached significance at 48h (Fig 4D).
Figure 4. Analysis of neutrophil activation in a one-step model of anti-GBM. A) Wild type mice were injected with anti-GBM and tissues were collected at different time points for staining of infiltrating neutrophils with Alexa-568(red) labelled secondary antibody and rat anti-mouse GR1, and overlaid with Hoechst nuclear stain (blue) B) Activation of neutrophils was further analysed with serum myeloperoxidase (MPO) measurement, and the impact on general inflammation with serum amyloid protein (SAP). C) Last, the impact of anti-GBM to complement was assessed with serum determination. All measurements were done in at least duplicate and significance was determined with One-Way ANOVA.
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Next we assessed the relationship between complement and neutrophils, where on the one hand activation products like C3a and C5a are potent chemoattractants for neutrophils, whereas neutrophils on the other hand have been described as an important cellular source of properdin [35]. Properdin was clearly present within the glomeruli 2h after anti-GM injection in WT mice.
However there was no clear co-localisation with GR1- positive neutrophils in the affected glomeruli. Neither properdin nor neutrophils were detectable after injection of control antibody (Fig 5A). The degree of neutrophil infiltration was not decreased in properdin-KO (Fig 5B), suggesting that in this model properdin-regulated AP activation of complement does not contribute to recruitment of neutrophils.
Figure 5. Colocalisation of neutrophil infiltration with properdin deposition in anti-GBM affected glomeruli. (A) The double staining of properdin and neutrophils (GR1) was performed 2h after anti-GBM administration. GR1 was detected with Alexa-568 labelled secondary antibody (red) and properdin with tyramide-FITC amplification (green). Deposition was evaluated in WT mice injected with control antibody, WT injected with anti-GBM
co-localisation of IgG and C3 changes over time
The rapid deposition of C3 in the glomerulus, combined with the systemic consumption of CP, suggested a CP-mediated activation mechanism. To verify this, we assessed co-localisation of Rb-IgG and C3 within glomeruli of WT mice injected with anti-GBM. There was clear linear co-localisation at early time points (2h – 6h), in line with the acute consumption of CP. However, this was followed by an increase of non-vascular C3 staining independent of IgG at 24-72h (Fig 6A). This suggests an alternative mode of C3 activation and deposition. Although we found clear co-deposition of properdin and C3 throughout the observation period, both for C3 and for properdin areas which contained only single deposits were also observed, especially at 48 – 72h when properdin stain was most prominent (Fig 6B).
Figure 6. Time course colocalization of Rabbit anti-GBM IgG with mouse C3, and mouse C3 with properdin (fP). Anti-GBM Rabbit IgG was detected with Alexa-488 (green) labelled secondary antibody and C3 with Alexa-568 (red) labelled secondary antibody, nuclei were stained with Hoechst (blue). Mouse Properdin was detected with HRP-labelled secondary antibody and Tyramide FITC amplification. All images were acquired with 40x amplification and representative glomerulus was chosen for comparison from stains performed on groups of four mice treated with anti-GBM at each time point.
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properdin can deposit in injured glomeruli independent of C3
Results above suggest that properdin might deposit in the injured glomerulus together with C3, but also independent of C3, as shown previously in vitro [17, 36]. However, the prominent presence of C3 makes it difficult to distinguish C3-independent properdin deposition. Therefore we studied properdin deposition in C3-KO mice 72 hr following anti-GBM treatment. We observed that properdin deposition was clearly present in anti-GBM-treated C3-KO mice and that most of the staining did not co-localise with the bound rabbit IgG from the anti-GBM (Fig 7). Specificity of this staining was confirmed by complete absence of properdin staining in anti-GBM-treated properdin- KO mice.
Figure 7. Co-localisation of properdin binding with C3 and rabbit IgG. (A) The deposition of properdin and rabbit IgG was analysed 72h after injection of anti-GBM into WT, C3KO and properdin KO mice. Properdin was detected with HRP labelled secondary antibody and Tyramide FITC amplification (green), and rabbit IgG with Alexa-568 (red) labelled secondary antibody (B) Co-staining of mouse C3 and properdin deposition in wild type mice at 72h after anti-
4. DISCUSSION
Recent studies have established that properdin has an interesting role in different disease pathologies, contributing to renal ischemia/reperfusion injury and possibly arthritis, but also being protective in C3 glomerulopahty and sepsis [24, 26, 31, 37]. Importantly, in vitro studies have suggested that properdin can specifically bind different ligands and initiate AP activation, independent of preceding C3 deposition, supporting the theory that properdin can act both as an initiation factor and as a positive regulator [9, 14, 16, 18, 20]. To better understand the mechanism of properdin binding and function in vivo, we established an experimental model of anti-GBM disease in wild type, properdin-deficient and C3-deficient mice. We demonstrate that also in vivo, properdin can act as a pattern recognition molecule and can deposit in injured glomeruli independent of C3.
For the purpose of analysing properdin and complement in experimental mouse models, we established and validated a novel properdin ELISA for analysis of serum properdin and a histological staining protocol for analysis of properdin and C9 deposition. Our results show that this model of anti- GBM resulted in an acute complement activation as shown by increased C3b/C3c/iC3b fragments 2h after injection. The role of properdin as a positive regulator of AP was further exemplified by the lower basal level and less prominent generation of C3 activation fragments in properdin KO mice [3, 4]. However, we did not observe an acute consumption of serum properdin in WT. In contrast we did find that serum properdin levels gradually increased until 72h after administration. We hypothesise that this could be in part due to limited local consumption within the glomeruli, and the fact that acute inflammation results in increased release of properdin into the circulation by activated inflammatory cells such as macrophages and neutrophils [35, 38].
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Functional analysis of plasma complement pathway activities were used to understand which pathways were involved in the acute complement activation.
The determinations were performed at the level of C3 activation, as we recently found that female mice have much lower level of serum terminal pathway (C5 – C9) activity (Kotimaa et al. submitted). WT mice showed uniform loss of CP and AP activities, and a heterogeneous consumption of LP, which supports the idea that the activation mechanism in anti-GBM is CP and AP mediated [28]. Interestingly, no consumption of CP and LP was observed in properdin- KO. In line with previous studies, the AP activity in properdin KO mice was not detectable as properdin is known to interact with LPS, the activation ligand in AP functional assays [14, 39]. Complement determinations did not reveal a marked gender difference in acute complement consumption.
Although properdin-KO and C3-KO reduced proteinuria by 30 – 37% compared to the WT mice, this protection did not reach significance. The female mice exhibited markedly higher proteinuria than male, which in part explains the heterogeneity of the responses to anti-GBM. The contribution of neutrophils to renal injury was then assessed, as these cells have been shown to contribute to the injury [28]. Our results revealed an acute and transient infiltration of neutrophils, with simultaneous increase of serum MPO and SAP. Together these results confirm prominent neutrophil activation contributing to the acute renal injury [40].
By sacrificing mice at different time points, we had the unique opportunity to investigate the natural course of complement activation at the tissue level.
First, properdin is known to be secreted by neutrophils upon activation, which has been thought to lead to local deposition at the site of activation [35, 41]. We found that properdin indeed is present in glomeruli already at 2h, however properdin staining did not clearly co-localise with neutrophil infiltrates. Therefore under these conditions the major source of properdin
show a reduced degree of neutrophil infiltrates, which would suggest that the CP-mediated C3 activation or anti-GBM immune-complexes alone are sufficient to recruit neutrophils to the glomeruli [28, 42]. To investigate the kinetics of complement activation, we analysed the deposition of C3, properdin and C9 between 2h and 72h. Our results showed an acute C3 and properdin deposition at 2h, which became more prominent at 24h – 72h. Interestingly C9 deposition was only weakly present at 24h, but continued to intensify from 48h – 72h, coinciding with increased C3 and properdin deposition. Together these results suggest that in this model of anti-GBM, the terminal pathway activation and resulting complement mediated renal injury and inflammation are attenuated until 48h – 72h [21, 43, 44].
Time course analysis of C3 and IgG co-localisation confirmed that initial complement activation is most likely CP dependent. However from 48h onwards C3 staining intensified and clearly became partially independent of anti-GBM IgG, coinciding with increased properdin and C9 deposition at 24 – 72h. Analysis of properdin and C3 clearly shows that the colocalisation becomes more prominent only after 48h. These results suggested that properdin may be binding to the glomeruli independent of CP mediated C3 deposition.
To confirm this finding, properdin deposition in anti-GBM injected C3-KO mice, which clearly demonstrated properdin deposition independent of C3.
Furthermore, the properdin deposition did not co-localise prominently with IgG, making it unlikely that deposited immune complexes would act as a ligand for properdin [45]. Although our study did not characterise the target of properdin binding in the injured glomeruli, it is tempting to speculate that this could be apoptotic and necrotic cells, to which properdin has been shown to bind [17, 36, 46].
In conclusion, using novel methods for analysis of properdin and other complement factors in experimental mouse models, we have demonstrated for the first time that properdin can bind to sites of injury in vivo independent of
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C3. These results support the in vitro findings that have suggested a properdin- directed model of AP activation [9, 14]. Furthermore, our model exhibits all the classical hallmarks of anti-GBM, which include systemic complement consumption, activation of AP and CP pathways, deposition of complement factors in the glomeruli, neutrophil activation, inflammation and severe renal injury [22, 28, 29, 47, 48].
These findings might be of interest for various AP and complement system- mediated diseases, as recent studies have established a role for properdin in C3 glomerulopathies [37, 49, 50]. Although AP has been shown to be a major contributor in experimental mouse renal ischemia/reperfusion injury, so far the role of properdin has only been determined DAF/CD59 double knockout mice, which are particularly sensitive for complement-mediated injury [24, 51]. The ability of properdin to interact independent of C3 in vivo expands its possible roles in diseases and injuries. In particular, the role of properdin in removal of apoptotic material [17, 36] and in neutrophil mediated diseases [16, 52–54] can be expanded further with future in vivo studies using the methods and reagents described here. Future studies with the acute model used here, and the attenuated anti-GBM used previously [28], would benefit from further time course analysis of renal injury with C3 KO, C4 KO and properdin KO mice, with different doses and strategies for disease induction. Furthermore, therapeutic depletion of properdin could be an attractive approach due to its protective impact on renal injury and disease, and should be evaluated in the context of anti-GBM as well [24, 55]. These studies would be integral to understand the role of complement-dependent and -independent injury in the context of anti-GBM disease.
acknowledgements
This work was financially supported in part by the European Union (Marie Curie TranSVIR FP7-PEOPLE-ITN-2008 No. 238756), a consortium grant from the Dutch Kidney Foundation (COMBAT) and performed in collaboration with EU FP7 project DIREKT (GA 602699).
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E415–22.
control WT fP KO C3 KO
n (female) 6 5 5
n (male) 4 3 4
n (all) 5 10 8 9
U-ACR
WT fP KO C3 KO
female 8.63 ± 3.7 4.4 ± 1.61 6.05 ± 2.14
male 4.24 ± 0.96 4.22 ± 3.02 3.25 ± 0.78
all 0.14 ± 0.03 6.87 ± 3.6 4.34 ± 2.02 4.81 ± 2.17
albumin
WT fP KO C3 KO
female 16346 ± 8805 10273 ± 6695 9370 ± 3229
male 7388 ± 860 5117 ± 2428 4600 ± 1439
all 12763 ± 7491 8340 ± 5867 7250 ± 3509
creatinine
WT fP KO C3 KO
female 2173 ± 1592 2304 ± 979 1575 ± 304
male 1808 ± 459 1409 ± 414 1408 ± 212
all 2027 ± 1150 1968 ± 901 1501 ± 266
