Properdin binds independent of complement activation in an in vivo model of anti–glomerular basement membrane disease
2018; Elsevier BV; Volume: 94; Issue: 6 Linguagem: Inglês
10.1016/j.kint.2018.06.030
ISSN1523-1755
AutoresJ. Dermot O’Flynn, Juha Kotimaa, Ria Faber-Krol, Karin Koekkoek, Ngaisah Klar‐Mohamad, Angela Koudijs, Wilhelm Schwaeble, Cordula Stover, Mohamed R. Daha, Cees van Kooten,
Tópico(s)Vasculitis and related conditions
ResumoProperdin is the only known positive regulator of complement activation by stabilizing the alternative pathway convertase through C3 binding, thus prolonging its half-life. Recent in vitro studies suggest that properdin may act as a specific pattern recognition molecule. To better understand the role of properdin in vivo, we used an experimental model of acute anti-glomerular basement membrane disease with wild-type, C3- and properdin knockout mice. The model exhibited severe proteinuria, acute neutrophil infiltration and activation, classical and alternative pathway activation, and progressive glomerular deposition of properdin, C3 and C9. Although the acute renal injury was likely due to acute neutrophil activation, we found properdin deposition in C3-knockout mice that was not associated with IgG. Thus, properdin may deposit in injured tissues in vivo independent of its main ligand C3. Properdin is the only known positive regulator of complement activation by stabilizing the alternative pathway convertase through C3 binding, thus prolonging its half-life. Recent in vitro studies suggest that properdin may act as a specific pattern recognition molecule. To better understand the role of properdin in vivo, we used an experimental model of acute anti-glomerular basement membrane disease with wild-type, C3- and properdin knockout mice. The model exhibited severe proteinuria, acute neutrophil infiltration and activation, classical and alternative pathway activation, and progressive glomerular deposition of properdin, C3 and C9. Although the acute renal injury was likely due to acute neutrophil activation, we found properdin deposition in C3-knockout mice that was not associated with IgG. Thus, properdin may deposit in injured tissues in vivo independent of its main ligand C3. The complement system consists of 3 activation pathways, classical (CP), lectin (LP), and alternative (AP), which converge at the level of C3 activation, proceeding to C5 cleavage and initiating the terminal pathway C5–C9 activation. Both the CP and LP have pattern recognition molecules (PRM), such as C1q and MBL, which in combination with the associated proteases initiate the complement cascade.1Walport M.J. Complement. First of two parts.N Engl J Med. 2001; 344: 1058-1066Crossref PubMed Scopus (2388) Google Scholar AP has an important role in augmenting CP and LP; once C3 is activated, the AP amplification loop enhances the complement cascade activation.2Camous L. Roumenina L. Bigot S. et al.Complement alternative pathway acts as a positive feedback amplification of neutrophil activation.Blood. 2011; 117: 1340-1349Crossref PubMed Scopus (156) Google Scholar, 3Harboe M. Ulvund G. Vien L. et al.The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation.Clin Exp Immunol. 2004; 138: 439-446Crossref PubMed Scopus (226) Google Scholar In addition, there is a constant low-level auto-activation of C3 (tick-over), which allows C3b formation on surfaces of pathogens and injured host cells.1Walport M.J. Complement. First of two parts.N Engl J Med. 2001; 344: 1058-1066Crossref PubMed Scopus (2388) Google Scholar, 4Kim D.D. Song W.C. Membrane complement regulatory proteins.Clin Immunol. 2006; 118: 127-136Crossref PubMed Scopus (261) Google Scholar The membrane-bound C3b associates with factor B, forming a short-lived AP C3 convertase C3bBb, which properdin binding stabilizes significantly, and making properdin the only positive regulator of complement.5Lutz H.U. Jelezarova E. Complement amplification revisited.Mol Immunol. 2006; 43: 2-12Crossref PubMed Scopus (66) Google Scholar, 6Fearon D.T. Austen K.F. Properdin: binding to C3b and stabilization of the C3b-dependent C3 convertase.J Exp Med. 1975; 142: 856-863Crossref PubMed Scopus (314) Google Scholar, 7Pillemer L. Blum L. Lepow I. et al.The properdin system and immunity. I. Demonstration and isolation of a new serum protein, properdin, and its role in immune phenomena.Science. 1954; 120: 279-285Crossref PubMed Scopus (406) Google Scholar, 8Kemper C. Atkinson J.P. Hourcade D.E. Properdin: emerging roles of a pattern-recognition molecule.Annu Rev Immunol. 2010; 28: 131-155Crossref PubMed Scopus (176) Google Scholar Recent in vitro studies have shown that properdin may exhibit PRM activity. Studies have shown targeted complement-fixing activity on certain ligands such as lipopolysaccharides (LPS),9Spitzer D. Mitchell L.M. Atkinson J.P. Hourcade D.E. Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly.J Immunol. 2007; 179: 2600-2608Crossref PubMed Scopus (229) Google Scholar oxidized LDL,10Klop B. van der Pol P. van Bruggen R. et al.Differential complement activation pathways promote C3b deposition on native and acetylated LDL thereby inducing lipoprotein binding to the complement receptor 1.J Biol Chem. 2014; 289: 35421-35430Crossref PubMed Scopus (17) Google Scholar and myeloperoxidase.11O'Flynn J, LUMC. Properdin-dependent activation and control of immune-homeostasis and autoimmunity. Available at: https://openaccess.leidenuniv.nl/handle/1887/28275. Accessed December 17, 2014.Google Scholar Similar results have been demonstrated with apoptotic and necrotic cells, and heparan sulfates expressed at the surface of renal epithelial cells.12Xu W. Berger S.P. Trouw L a et al.Properdin binds to late apoptotic and necrotic cells independently of C3b and regulates alternative pathway complement activation.J Immunol. 2008; 180: 7613-7621Crossref PubMed Scopus (124) Google Scholar, 13Gaarkeuken H. Siezenga M.A. Zuidwijk K. et al.Complement activation by tubular cells is mediated by properdin binding.Am J Physiol Renal Physiol. 2008; 295: F1397-F1403Crossref PubMed Scopus (68) Google Scholar, 14Zaferani A. Vivès R.R. van der Pol P. et al.Identification of tubular heparan sulfate as a docking platform for the alternative complement component properdin in proteinuric renal disease.J Biol Chem. 2011; 286: 5359-5367Crossref PubMed Scopus (49) Google Scholar, 15Zaferani A. Vivès R.R. van der Pol P. et al.Factor h and properdin recognize different epitopes on renal tubular epithelial heparan sulfate.J Biol Chem. 2012; 287: 31471-31481Crossref PubMed Scopus (41) Google Scholar, 16Kemper C. Mitchell L.M. Zhang L. Hourcade D.E. The complement protein properdin binds apoptotic T cells and promotes complement activation and phagocytosis.Proc Natl Acad Sci U S A. 2008; 105: 9023-9028Crossref PubMed Scopus (129) Google Scholar In this model properdin attaches to specific ligands and forms stable C3bBbP convertase, possibly overcoming the local threshold for C5 convertase (C3bBbC3b) formation.8Kemper C. Atkinson J.P. Hourcade D.E. Properdin: emerging roles of a pattern-recognition molecule.Annu Rev Immunol. 2010; 28: 131-155Crossref PubMed Scopus (176) Google Scholar, 9Spitzer D. Mitchell L.M. Atkinson J.P. Hourcade D.E. Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly.J Immunol. 2007; 179: 2600-2608Crossref PubMed Scopus (229) Google Scholar, 12Xu W. Berger S.P. Trouw L a et al.Properdin binds to late apoptotic and necrotic cells independently of C3b and regulates alternative pathway complement activation.J Immunol. 2008; 180: 7613-7621Crossref PubMed Scopus (124) Google Scholar However, it is not yet clear how properdin could act as a PRM, as it lacks known recognition domains.17Pedersen D.V. Roumenina L. Jensen R.K. et al.Functional and structural insight into properdin control of complement alternative pathway amplification.EMBO J. 2017; 36: 1084-1099Crossref PubMed Scopus (45) Google Scholar AP and properdin have been shown to contribute to arthritis, abdominal aortic aneurysm, and renal ischemia reperfusion injury (I/RI). However, these studies have not been able to differentiate whether the deposition of properdin in tissues takes place via membrane-bound C3b or through epitopes present in the injured tissues.18Miwa T. Sato S. Gullipalli D. et al.Blocking properdin, the alternative pathway, and anaphylatoxin receptors ameliorates renal ischemia-reperfusion injury in decay-accelerating factor and CD59 double-knockout mice.J Immunol. 2013; 190: 3552-3559Crossref PubMed Scopus (55) Google Scholar, 19Kimura Y. Zhou L. Miwa T. Song W.C. Genetic and therapeutic targeting of properdin in mice prevents complement-mediated tissue injury.J Clin Invest. 2010; 120: 3545-3554Crossref PubMed Scopus (59) Google Scholar, 20Dimitrova P. Ivanovska N. Schwaeble W. et al.The role of properdin in murine zymosan-induced arthritis.Mol Immunol. 2010; 47: 1458-1466Crossref PubMed Scopus (37) Google Scholar, 21Zhou H.H.-F. Yan H. Stover C.M.C. et al.Antibody directs properdin-dependent activation of the complement alternative pathway in a mouse model of abdominal aortic aneurysm.Proc Natl Acad Sci U S A. 2012; 109: E415-E422Crossref PubMed Scopus (57) Google Scholar To study the sequence of properdin binding in vivo, we established an acute model of anti–glomerular basement membrane (GBM) disease in wild-type (WT), C3 knockout (KO), and properdin KO mice. Recent studies on anti-GBM disease have shown that properdin and other AP components are prominently present in anti-GBM–affected glomeruli in humans and that AP contributes to the injury in mice,22Ma R. Cui Z. Liao Y.H. Zhao M.H. Complement activation contributes to the injury and outcome of kidney in human anti-glomerular basement membrane disease.J Clin Immunol. 2013; 33: 172-178Crossref PubMed Scopus (32) Google Scholar, 23Ma R. Cui Z. Hu S.-Y. et al.The alternative pathway of complement activation may be involved in the renal damage of human anti-glomerular basement membrane disease.PLoS One. 2014; 9: e91250Crossref PubMed Scopus (31) Google Scholar, 24Otten M a Groeneveld T.W.L. Flierman R. et al.Both complement and IgG fc receptors are required for development of attenuated antiglomerular basement membrane nephritis in mice.J Immunol. 2009; 183: 3980-3988Crossref PubMed Scopus (32) Google Scholar making the model attractive for studying properdin. Acute complement activation was evaluated with comparison of mouse plasma before and 2 hours post-administration of anti-GBM antibody, showing significantly increased C3b/C3c/iC3b in WT mice (3.6-fold), which was not seen following administration of control IgG or in properdin-KO (fP KO) mice (Figure 1a). Moreover, basal levels of C3 activation fragments was 2-fold lower in fP KO mice compared with the WT mice. The novel mouse properdin enzyme-linked immunosorbent assay (ELISA) has a linear detection range with recombinant mouse properdin (Figure 1b) with no detectable properdin in fP KO mouse serum (Figure 1c). No changes in circulating properdin were found 2 hours after injection of WT mice with anti-GBM or control antibody (Figure 1c). Functional ELISAs established intact serum complement in WT mice and reduced AP activity in the fP KO mice. Two hours after anti-GBM antibody injection, prominent consumption of CP (Figure 1d) and AP (Figure 1e) was seen, whereas findings for LP consumption were inconclusive (Figure 1e). No consistent complement consumption was observed with control antibody–injected mice. In properdin KO mice anti-GBM antibody injection did not result in significant CP or LP pathway consumption (Figure 1f). Together these results show prominent complement activation in this anti-GBM model, with partial dependence on properdin. The kinetics of renal complement activation following anti-GBM antibody injection were evaluated in groups of WT mice killed at different time points. The anti-GBM rabbit IgG (Rb IgG) was clearly present in glomerular vasculature from 2 to 72 hours (Figure 2). The C1q staining was performed as a marker of CP involvement, showing an acute and unchanged staining pattern from 2 to 72 hours, closely resembling that of Rb IgG (Figure 2). The C3 deposition was present in glomeruli at 2 hours and increased until 72 hours. The C3 deposition closely resembled the pattern of rabbit IgG until 48 hours, after which a focal staining pattern became more apparent (Fig 2). Properdin deposition was present at 2 hours, with clear increase from 24 hours onwards. Interestingly, properdin (fP) staining did not exhibit the linear pattern found with IgG and C3 (Figure 2). The staining for both C6 and C9 exhibited late kinetics, emerging only after 24 hours and becoming most prominent at 48 hours (Figure 2). In all cases specificity of the staining was confirmed with isotype and biological positive and negative controls (Supplementary Figure S1). We did not observe gender-specific differences in staining pattern or intensity in any of the studied complement factors (data not shown). The rapid deposition of C3 in the glomerulus, combined with the systemic consumption of CP and linear deposition of both Rb IgG and C1q, suggested an IgG- and CP-mediated activation mechanism. To verify this, we assessed glomerular colocalization of Rb IgG and C3 with anti-GBM antibody–injected WT mice. The time-course histology showed that the deposition of C3 closely resembles both rabbit IgG and C1q staining in the glomerular endothelium at 2 to 6 hours, with a clearly defined vascular loop structure. From 48 hours onwards, the C3 deposits lining the vascular endothelium become more prominent, and significant C3 staining is present in the lumen independent from rabbit IgG deposits (Figure 3a). With the same mice, we found clear colocalization of properdin and C3 throughout the observation period, suggestive of AP-mediated C3 activation (Figure 3b). However, the single deposits of both C3 and properdin were also observed, especially at 48 to 72 hours, when properdin staining was most prominent (Figure 3b). Properdin deposition in C3-KO mice was demonstrated at various time points (2, 24, and 72 hours) following anti-GBM antibody treatment (Figure 4a). Colocalization of rabbit IgG with properdin was only incidental, and properdin exhibited strong single staining in both WT and C3 KO mice, with no evident staining in fP KO mice (Figure 4b). To investigate cell death, we performed terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in 72-hour biopsies (Figure 4c). TUNEL assay positivity was observed in glomeruli staining positive for rabbit IgG, C3, and properdin. However, when looking in detail, especially for C3 and properdin, only a minor fraction showed colocalization (Figure 4c). Analysis of TUNEL and properdin staining in C3 KO mice showed close but nonoverlapping colocalization within glomeruli at 72 hours, and properdin staining was more abundant compared with TUNEL staining (Figure 4d). Next, we evaluated the impact of properdin KO on the anti-GBM antibody–induced renal injury using urine albumin-creatinine ratio (UACR) as a functional read-out (Figure 5). The injection of anti-GBM antibody in WT C57bl/6 resulted in severe albuminuria in 24-hour urine collected between 72 and 96 hours. The UACR in WT mice was significantly higher in anti-GBM antibody–injected mice compared with control antibody–injected mice (60,761 ± 30,011 mg/g and 1205 ± 282 mg/g, respectively). Compared with WT mice, the properdin KO and C3 KO mice showed a 36% to 40% lower level of albuminuria (UACR of 36,420 ± 16,994 mg/g and 40,373 ± 18247 mg/g, respectively). However, statistical difference was not reached. Female WT and C3 KO mice exhibited a higher degree of renal injury than did male mice. In properdin KO mice no gender differences were observed (Supplementary Table S1). Neutrophils have been shown to contribute to renal injury in experimental models of anti-GBM disease.24Otten M a Groeneveld T.W.L. Flierman R. et al.Both complement and IgG fc receptors are required for development of attenuated antiglomerular basement membrane nephritis in mice.J Immunol. 2009; 183: 3980-3988Crossref PubMed Scopus (32) Google Scholar Time-course analysis of neutrophils using a GR1 staining showed that infiltration within the glomeruli was biphasic, being most prominent at 2 hours, returning to low levels at 24 to 48 hours, and increasing at 72 hours (Figure 6a). Acute infiltration and activation of neutrophils was supported by a transient 3-fold increase in serum myeloperxodase (MPO) at 2 to 6 hours (Figure 6b), which was followed with serum amyloid protein (SAP) peaking at 24 hours and remaining elevated until 72 hours (Figure 6c). Serum properdin showed a gradual increase from baseline, which reached significance at 48 hours (Figure 6d). Complement anaphylatoxins C3a and C5a are potent chemoattractants for inflammatory cells. Additionally, neutrophils contribute to the systemic properdin pool and can secrete it locally.25Wirthmueller U. Dewald B. Thelen M. et al.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, 26Bao L. Wang Y. Haas M. Quigg R.J. Distinct roles for C3a and C5a in complement-induced tubulointerstitial injury.Kidney Int. 2011; 80: 524-534Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar There was no clear colocalization with properdin and GR1-positive neutrophils in the affected glomeruli at 2 hours, and neither properdin nor neutrophils were present in control antibody–treated mice (Figure 7a). Furthermore, the degree of neutrophil infiltration was not decreased in properdin KO mice (Figure 7b), suggesting that in this model properdin-regulated activation of complement does not contribute to recruitment of neutrophils. Recent studies have established that properdin has differing roles in disease pathologies, with a contributing role in experimental models of renal IRI and possibly arthritis and a protective role in C3 glomerulopathy and sepsis.18Miwa T. Sato S. Gullipalli D. et al.Blocking properdin, the alternative pathway, and anaphylatoxin receptors ameliorates renal ischemia-reperfusion injury in decay-accelerating factor and CD59 double-knockout mice.J Immunol. 2013; 190: 3552-3559Crossref PubMed Scopus (55) Google Scholar, 20Dimitrova P. Ivanovska N. Schwaeble W. et al.The role of properdin in murine zymosan-induced arthritis.Mol Immunol. 2010; 47: 1458-1466Crossref PubMed Scopus (37) Google Scholar, 27Ruseva M.M. Vernon K a Lesher A.M. et al.Loss of properdin exacerbates C3 glomerulopathy resulting from factor H deficiency.J Am Soc Nephrol. 2013; 24: 43-52Crossref PubMed Scopus (59) Google Scholar, 28Stover C.M. Luckett J.C. Echtenacher B. et al.Properdin plays a protective role in polymicrobial septic peritonitis.J Immunol. 2008; 180: 3313-3318Crossref PubMed Scopus (68) Google Scholar The role of properdin as a positive regulator of AP is clear. However, the possible PRM role remains unsolved. Recent in vitro studies show that properdin has PRM activity and can act as a focus point of local C3b deposition on several ligands.8Kemper C. Atkinson J.P. Hourcade D.E. Properdin: emerging roles of a pattern-recognition molecule.Annu Rev Immunol. 2010; 28: 131-155Crossref PubMed Scopus (176) Google Scholar, 9Spitzer D. Mitchell L.M. Atkinson J.P. Hourcade D.E. Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly.J Immunol. 2007; 179: 2600-2608Crossref PubMed Scopus (229) Google Scholar, 13Gaarkeuken H. Siezenga M.A. Zuidwijk K. et al.Complement activation by tubular cells is mediated by properdin binding.Am J Physiol Renal Physiol. 2008; 295: F1397-F1403Crossref PubMed Scopus (68) Google Scholar, 15Zaferani A. Vivès R.R. van der Pol P. et al.Factor h and properdin recognize different epitopes on renal tubular epithelial heparan sulfate.J Biol Chem. 2012; 287: 31471-31481Crossref PubMed Scopus (41) Google Scholar, 29O'Flynn J. Dixon K.O. Faber Krol M.C. Daha M.R. Van Kooten C. Myeloperoxidase directs properdin-mediated complement activation.J Innate Immun. 2014; 6: 417-425Crossref PubMed Scopus (41) Google Scholar Conversely, in other cases properdin has been shown to act only as a positive regulator, requiring preceding C3b deposition to bind.30Harboe M. Garred P. Lindstad J.K. et al.The role of properdin in zymosan- and Escherichia coli-induced complement activation.J Immunol. 2012; 189: 2606-2613Crossref PubMed Scopus (38) Google Scholar, 31Harboe M. Johnson C. Nymo S. et al.Properdin binding to complement activating surfaces depends on initial C3b deposition.Proc Natl Acad Sci U S A. 2017; 114: E534-E539Crossref PubMed Scopus (48) Google Scholar To better understand the mechanism of properdin binding and function in vivo, we explored an experimental model of anti-GBM disease in WT, properdin KO, and C3 KO mice. Our model exhibited typical characteristics of an experimental anti-GBM disease, 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.23Ma R. Cui Z. Hu S.-Y. et al.The alternative pathway of complement activation may be involved in the renal damage of human anti-glomerular basement membrane disease.PLoS One. 2014; 9: e91250Crossref PubMed Scopus (31) Google Scholar, 24Otten M a Groeneveld T.W.L. Flierman R. et al.Both complement and IgG fc receptors are required for development of attenuated antiglomerular basement membrane nephritis in mice.J Immunol. 2009; 183: 3980-3988Crossref PubMed Scopus (32) Google Scholar, 32Sheerin N.S. Springall T. Carroll M.C. et al.Protection against anti-glomerular basement membrane (GBM)-mediated nephritis in C3- and C4-deficient mice.Clin Exp Immunol. 1997; 110: 403-409Crossref PubMed Scopus (102) Google Scholar, 33Groggel G.C. Adler S. Rennke H.G. et al.Role of the terminal complement pathway in experimental membranous nephropathy in the rabbit.J Clin Invest. 1983; 72: 1948-1957Crossref PubMed Scopus (66) Google Scholar, 34Boyce N.W. Holdsworth S.R. Anti-glomerular basement membrane antibody-induced experimental glomerulonephritis: evidence for dose-dependent, direct antibody and complement-induced, cell-independent injury.J Immunol. 1985; 135: 3918-3921PubMed Google Scholar Acute complement activation was verified with plasma C3b/C3c/iC3b fragments at 2 hours, which also revealed the impact of properdin as a positive regulator of AP because properdin KO mice had a lower basal level of C3 activation fragments and less prominent generation of C3 fragments. Furthermore, we observed acute consumption of CP and AP, which was not present in properdin KO mice, underlining the role of the properdin and AP amplification loop.5Lutz H.U. Jelezarova E. Complement amplification revisited.Mol Immunol. 2006; 43: 2-12Crossref PubMed Scopus (66) Google Scholar Interestingly, we did not observe serum properdin consumption in WT mice. Conversely, the levels increased in tandem with SAP, suggesting that systemic inflammation may affect the secretion of properdin to circulation from activated inflammatory cells.25Wirthmueller U. Dewald B. Thelen M. et al.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, 35Schwaeble W. Huemer H.P. Möst J. et al.Expression of properdin in human monocytes.Eur J Biochem. 1994; 219: 759-764Crossref PubMed Scopus (47) Google Scholar, 36Dixon K.O. O'Flynn J. Klar-Mohamad N. Daha M.R. van Kooten C. Properdin and factor H production by human dendritic cells modulates their T-cell stimulatory capacity and is regulated by IFN-γ.Eur J Immunol. 2017; 47: 470-480Crossref PubMed Scopus (19) Google Scholar It must be noted that the range of mouse properdin seemed high compared with concentrations in human serum. This may be due to the use of recombinant mouse properdin as a standard that may not have the same distribution of properdin isoforms as is found in vivo.37Stover C.M. McDonald J. Byrne S. et al.Properdin levels in human sepsis.Front Immunol. 2015; 6: 24Crossref PubMed Scopus (13) Google Scholar Time-course immunohistochemistry revealed an acute C1q, C3, and properdin deposition in line with serum determinations, suggesting CP- and AP-mediated complement activation. However, C6 and C9 deposition became evident only after 24 hours and continued to intensify from 48 to 72 hours, coinciding with increased C3 and properdin deposition. Together, these results suggest that in this model of anti-GBM disease, the complement-mediated injury may be attenuated until 48 to 72 hours. Further analysis of C3 colocalization confirmed that initial complement activation is most likely CP dependent and is present along the vascular endothelium. However, after 48 hours the C3 staining intensified and colocalized more with properdin, suggesting AP and properdin involvement in complement activation. We demonstrated that properdin deposition was present in glomeruli of anti-GBM–injected C3 KO mice. We showed some colocalization of properdin with TUNEL-positive cells, potentially confirming interaction with apoptotic cells.12Xu W. Berger S.P. Trouw L a et al.Properdin binds to late apoptotic and necrotic cells independently of C3b and regulates alternative pathway complement activation.J Immunol. 2008; 180: 7613-7621Crossref PubMed Scopus (124) Google Scholar, 38Banda N.K. Takahashi K. Wood A.K. et al.Pathogenic complement activation in collagen antibody-induced arthritis in mice requires amplification by the alternative pathway.J Immunol. 2007; 179: 4101-4109Crossref PubMed Scopus (90) Google Scholar However, a large part of the properdin was independent of TUNEL, thereby indicating the presence of other, yet unknown ligands. Properdin KO and C3 KO resulted in reduced proteinuria compared with the WT mice; however, this protection was not statistically significant. This is in contrast to previous studies with a 2-step model, which established involvement of both complement and neutrophils in the experimental renal injury in male mice.24Otten M a Groeneveld T.W.L. Flierman R. et al.Both complement and IgG fc receptors are required for development of attenuated antiglomerular basement membrane nephritis in mice.J Immunol. 2009; 183: 3980-3988Crossref PubMed Scopus (32) Google Scholar Although small group size did not allow for proper statistical analysis, female mice seemed to exhibit markedly higher proteinuria in WT and C3 KO mice in this model. We did not observe differences in complement stains between female and male mice, including C6 and C9. We have recently shown that female C57bl/6 mice have significantly reduced terminal complement pathway functionality.39Kotimaa J. Klar-Mohammad N. Gueler F. et al.Sex matters: Systemic complement activity of female C57BL/6J and BALB/cJ mice is limited by serum terminal pathway components.Mol Immunol. 2016; 76: 13-21Crossref PubMed Scopus (47) Google Scholar In light of our results here, we can conclude that this residual complement activity results in prominent complement deposition in vivo when the injury is localized, such as in the glomeruli. Our results show an acute and transient infiltration of neutrophils, with simultaneous increase of serum MPO and SAP that precedes the full complement activation. Furthermore, properdin KO did not impact neutrophil infiltration, and no colocalization between neutrophils and properdin was observed. Together our results show that in this model the initial CP- and AP-mediated C3 activation and anti-GBM IgG are sufficient to recruit neutrophils to the glomeruli together with the rabbit IgG Fc receptor. Our results show only limited amelioration of renal injury using C3 KO mice, suggesting that the neutrophils are the likely driver of the acute injury, and that the full complement activation dependent on CP, AP, and properdin is attenuated and may become a contributing factor later.24Otten M a Groeneveld T.W.L. Flierman R. et al.Both complement and IgG fc receptors are required for development of attenuated antiglomerular basement membrane nephritis in mice.J Immunol. 2009; 183: 3980-3988Crossref PubMed Scopus (32) Google Scholar, 40Coxon A. Cullere X. Knight S. et al.FcγRIII Mediates Neutrophil Recruitment to Immune Complexes.Immunity. 2001; 14: 693-704Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar Taken together, our results show that properdin is present in injured glomeruli in vivo, independent of C3 involvement. The mechanism and putative ligand facilitating the deposition remains unclear, as only incidental colocalization was found with C3, TUNEL-positive cells, neutrophils, and Igs. Further studies should aim to identify whether this finding is a result of specific epitopes or passive entrapment within the injured glomeruli, and whether deposition of properdin may direct further C3 activation in vivo. These findings are interesting for various AP- and complement-mediated diseases. 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 material12Xu W. Berger S.P. Trouw L a et al.Properdin binds to late apoptotic and necrotic cells independently of C3b and regulates alternative pathway complement activation.J Immunol. 2008; 180: 7613-7621Crossref PubMed Scopus (124) Google Scholar, 16Kemper C. Mitchell L.M. Zhang L. Hourcade D.E. The complement protein properdin binds apoptotic T cells and promotes complement activation and phagocytosis.Proc Natl Acad Sci U S A. 2008; 105: 9023-9028Crossref PubMed Scopus (129) Google Scholar and in neutrophil-mediated diseases29O'Flynn J. Dixon K.O. Faber Krol M.C. Daha M.R. Van Kooten C. Myeloperoxidase directs properdin-mediated complement activation.J Innate Immun. 2014; 6: 417-425Crossref PubMed Scopus (41) Google Scholar, 41Xiao H. Schreiber A. Heeringa P. et al.Alternative complement pathway in the pathogenesis of disease mediated by anti-neutrophil cytoplasmic autoantibodies.Am J Pathol. 2007; 170: 52-64Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 42Gou S.-J. Yuan J. Chen M. et al.Circulating complement activation in patients with anti-neutrophil cytoplasmic antibody–associated vasculitis.Kidney Int. 2012; 83: 129-137Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 43Xing G.Q. Chen M. Liu G. et al.Complement activation is involved in renal damage in human antineutrophil cytoplasmic autoantibody associated pauci-immune vasculitis.J Clin Immunol. 2009; 29: 282-291Crossref PubMed Scopus (160) Google Scholar can be expanded further with future in vivo studies using the methods, animals, and reagents described here. C57BL/6 WT mice were purchased from Charles River Laboratories (Wilmington, MA). C3 KO mice were a kind gift from Mike Carroll (Harvard Medical School, Boston, MA), and properdin KO mice were maintained by Cordula Stover (University of Leicester, Leicester, UK).28Stover C.M. Luckett J.C. Echtenacher B. et al.Properdin plays a protective role in polymicrobial septic peritonitis.J Immunol. 2008; 180: 3313-3318Crossref PubMed Scopus (68) Google Scholar All experiments were approved by the Leiden University animal ethical committee and performed according to institutional and national guidelines. Rabbit anti-rat GBM IgG, cross-reacting with mouse GBM, was prepared in house as previously described,44Joosten S.A. van Dixhoorn M.G.A. Borrias M.C. et al.Antibody response against perlecan and collagen types IV and VI in chronic renal allograft rejection in the rat.Am J Pathol. 2002; 160: 1301-1310Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar and preparation and cross-reactivity were confirmed in pilot experiments with anti-GBM and control antibodies injected into WT C57BL/6 mice. For the model, both male and female age-matched (range: 7–10 weeks old) mice (C57BL/6 WT, C3-KO, and properdin KO mice) were used. At day 0, the mice were injected i.v. with rabbit anti-GBM or control rabbit IgG (0.5 mg in 200 μl physiological saline). Acute anti-GBM disease was evaluated at 2 hours after injection. WT mice were injected with anti-GBM or control antibody, whereas properdin KO mice were injected only with anti-GBM antibody. For complement determinations, ethylenediamine tetra-acetic acid plasma was collected by tail cuts from each mouse before and 2 hours after injection without anesthesia. Serum was collected after CO2 killing via heart puncture. All blood samples were placed directly on ice and prepared as plasma or serum as described previously.45Kotimaa J.J.P. van Werkhoven M.B.M.B. O'Flynn J. et al.Functional assessment of mouse complement pathway activities and quantification of C3b/C3c/iC3b in an experimental model of mouse renal ischaemia/reperfusion injury.J Immunol Methods. 2015; 419: 25-34Crossref PubMed Scopus (18) Google Scholar 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 24-hour urine collection and proteinuria analysis from anti-GBM antibody–injected WT (n = 10), properdin-KO (n = 8), and C3 KO (n = 9) mice and WT mice injected with control antibody (n = 5). For urine collection mice were placed in metabolic cages 72 hours after anti-GBM antibody administration with free access to food and water. The collected urine was centrifuged, aliquoted, and stored at –20°C. The kinetics of complement and neutrophil activation were studied with groups of WT mice killed at 2 to 72 hours after injection of anti-GBM IgG. Additional kidneys were collected at 2 hours from properdin KO mice, and at 72 hours from C3 KO mice that were injected with either anti-GBM or control IgG. The mice were killed with CO2, and kidneys were harvested and snap-frozen for later histological analysis. Urine albumin was quantified using rocket immunoelectrophoresis as previously described.24Otten M a Groeneveld T.W.L. Flierman R. et al.Both complement and IgG fc receptors are required for development of attenuated antiglomerular basement membrane nephritis in mice.J Immunol. 2009; 183: 3980-3988Crossref PubMed Scopus (32) Google Scholar Urine creatinine was quantified with creatinine strips for the Reflotron Plus system (Roche Diagnostics, Risch-Rotkreuz, Switzerland). The degree of proteinuria was calculated as the UACR. A specific ELISA for mouse properdin was developed with mouse anti-mouse properdin monoclonal antibody (clone 17-17) and rabbit polyclonal antibody raised against recombinant mouse properdin.46Ali Y.M. Hayat A. Saeed B.M. et al.Low-dose recombinant properdin provides substantial protection against Streptococcus pneumoniae and Neisseria meningitidis infection.Proc Natl Acad Sci U S A. 2014; 111: 5301-5306Crossref PubMed Scopus (40) Google Scholar The capture monoclonal antibody was coated at 2 μg/ml in 96-well ELISA plates (Nunc Maxisorp; Thermo Fisher Scientific, Waltham, MA). Following coating, the wells were blocked with 1% bovine serum albumin in phosphate buffered saline (PBS). Normal mouse CD-1 serum, recombinant mouse properdin, or properdin KO serum was serially diluted in PBS, 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO), 1% bovine serum albumin (phenacyl thiazolium bromide) and added to the blocked wells. Captured properdin was detected using digoxigenin (DIG)-labeled rabbit anti-human properdin (in house, Leiden University Medical Center), anti-DIG-POD (11207733910, Roche Diagnostics), followed by colorimetric quantification with a colorimetric substrate step with 3,3',5,5'-tetramethylbenzidine for 15 to 30 minutes at room temperature and stopped with 50 μl 1M H2SO4 and read at 450 nm with a Bio-Rad 550 instrument (Bio-Rad, Hercules, CA). Measurement of functional mouse complement pathway activities was performed with an ELISA-based system as described previously.39Kotimaa J. Klar-Mohammad N. Gueler F. et al.Sex matters: Systemic complement activity of female C57BL/6J and BALB/cJ mice is limited by serum terminal pathway components.Mol Immunol. 2016; 76: 13-21Crossref PubMed Scopus (47) Google Scholar, 45Kotimaa J.J.P. van Werkhoven M.B.M.B. O'Flynn J. et al.Functional assessment of mouse complement pathway activities and quantification of C3b/C3c/iC3b in an experimental model of mouse renal ischaemia/reperfusion injury.J Immunol Methods. 2015; 419: 25-34Crossref PubMed Scopus (18) Google Scholar In brief, human IgM-coated plates were used for CP, Mannan-coated plates for LP, and LPS-coated plates for AP ELISA. 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 Ca2+-chelating EGTA. Complement activity at the level of C3 was determined with biotinylated rat anti-mouse C3b-C3c-iC3b monoclonal antibody clone 2/1147Mastellos D. Prechl J.O. László G. et al.Novel monoclonal antibodies against mouse C3 interfering with complement activation: Description of fine specificity and applications to various immunoassays.Mol Immunol. 2004; 40: 1213-1221Crossref PubMed Scopus (55) Google Scholar (HM1065; Hycult Biotech, Uden, Netherlands) and Streptavidin-HRP conjugate (Hycult Biotech). Commercial assays were used to measure SAP (HK215, Hycult Biotech) and MPO (HK210, Hycult Biotech) according to manufacturer's specifications. The C3b/C3c/iC3b quantification was performed as described previously.45Kotimaa J.J.P. van Werkhoven M.B.M.B. O'Flynn J. et al.Functional assessment of mouse complement pathway activities and quantification of C3b/C3c/iC3b in an experimental model of mouse renal ischaemia/reperfusion injury.J Immunol Methods. 2015; 419: 25-34Crossref PubMed Scopus (18) Google Scholar Kidneys were sectioned into 4-μm slices using a cryostat, fixed by 10-minute incubation in acetone, and washed with PBS 3 times 5 minutes after each step, and all antibodies were diluted in 1% bovine serum albumin and PBS. Mouse properdin, C6, and C9 were detected using tyramide-fluorescein isothiocyanate (FITC) amplification–based staining. The slides were first treated with 45-minute room temperature incubation in PBS buffer containing 0.6% H2O2 (1.07209.0250; Merck KGaA, Darmstadt, Germany) and 0.2% NaN3 (1.06688.0100, Merck) diluted in PBS. Properdin was detected with 1 μg/ml diluted rabbit anti-mouse properdin-DIG described for the properdin ELISA (in house, Leiden University Medical Center), whereas mouse C6 and C9 were detected with 1 μg/ml rabbit anti-mouse recombinant C6-DIG and C9-DIG described previously.45Kotimaa J.J.P. van Werkhoven M.B.M.B. O'Flynn J. et al.Functional assessment of mouse complement pathway activities and quantification of C3b/C3c/iC3b in an experimental model of mouse renal ischaemia/reperfusion injury.J Immunol Methods. 2015; 419: 25-34Crossref PubMed Scopus (18) Google Scholar For both components, the detection was followed with overnight incubation of the slides with 1/750 diluted sheep anti-DIG-POD (Roche Diagnostics). Finally, the slides were incubated 20 minutes with 1/500 diluted tyramide-FITC (T9034-4, Sigma-Aldrich) in tyramide buffer (NENTM, Life Science Products, Chestertown, MD) with 0.01% H2O2 (Merck). Mouse GR1 was detected with 1/400 diluted rat anti-mouse GR1 (a kind gift from Georg Kraal, VU University Medical Center, Amsterdam, Netherlands) and 1/750 diluted goat anti-rat-Alexa 568 (A11011; Molecular Probes, Eugene, OR). Deposition of rabbit IgG to mouse glomeruli was detected with 1/400 diluted goat anti-rabbit IgG-Alexa 488 (A11008, Molecular Probes) or 1/400 diluted goat anti-rabbit IgG-Alexa568 (ab175471; Abcam, Cambridge, UK). 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, Carlsbad, CA) according to the manufacturer's instructions. Fluorescence microscopy was performed with a Zeiss Axio Scope A1 with an EX-plan Neofluar 40x lens and captured with Axiocam MRC5 (Carl Zeiss, Dublin, CA). Specificity of the staining was confirmed with renal tissue harvested 72 hours after injection of antibody. Rabbit IgG and C9 stain specificity was controlled with mice injected with control rabbit IgG. Properdin and C3 staining specificity was tested with properdin KO and C3 KO mice injected with anti-GBM antibody. In all cases the staining was free of nonspecific signal within the glomeruli, as assessed by isotype controls. All the authors declared no competing interests. This work was financially supported in part by the European Union (Marie Curie TranSVIR FP7-PEOPLE-ITN-2008 no. 238756) and by a Consortium grant from the Dutch Kidney Foundation (COMBAT, the Netherlands). Download .docx (.01 MB) Help with docx files Table S1Analysis of proteinuria and impact of gender to renal injury. Table depicts the urinary albumin-creatinine ratio (UACR), urinary albumin (μg/ml) and creatinine (μmol/l). The 24-hour urine was collected between 72 to 96 hours in individual metabolic cages from wild-type C57bl/6 (WT), properdin (fP) knockout (KO), and C3 KO mice. Values are depicted as mean ± SD. Download .docx (.5 MB) Help with docx files Figure S1Specificity of renal histology stains. (A) The specificity of properdin (fP) and C3 stains were assessed with fP knockout (KO) and C3 KO mice, respectively, and compared against wild-type (WT) mice. The mice were injected with 0.5 mg rabbit (Rb) IgG anti–glomerular basement membrane (GBM) antibody, and tissues were harvested at 72 hours. (B) Specificity of Rb IgG, C9, and GR1 stains were evaluated against tissues collected from WT mice injected with either 0.5 mg nonspecific Rb IgG or 0.5 mg Rb IgG anti-GBM. Mouse C9 assessment was performed on tissues collected at 96 hours after injection, whereas GR1 and Rb IgG were assessed with tissues collected at 2 hours after injection.
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