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Blocking Properdin, the Alternative Pathway, and Anaphylatoxin Receptors Ameliorates Renal Ischemia-Reperfusion Injury in Decay-Accelerating Factor and CD59 This information is current as Double-Knockout Mice of September 26, 2021. Takashi Miwa, Sayaka Sato, Damodar Gullipalli, Masaomi Nangaku and Wen-Chao Song J Immunol published online 20 February 2013 http://www.jimmunol.org/content/early/2013/02/20/jimmun Downloaded from ol.1202275 http://www.jimmunol.org/ Why The JI? Submit online.

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 20, 2013, doi:10.4049/jimmunol.1202275 The Journal of Immunology

Blocking Properdin, the Alternative Pathway, and Anaphylatoxin Receptors Ameliorates Renal Ischemia-Reperfusion Injury in Decay-Accelerating Factor and CD59 Double-Knockout Mice

Takashi Miwa,*,† Sayaka Sato,*,† Damodar Gullipalli,*,† Masaomi Nangaku,‡ and Wen-Chao Song*,†

Complement is implicated in the pathogenesis of ischemia-reperfusion injury (IRI). The activation pathway(s) and effector(s) of complement in IRI may be organ speciﬁc and remain to be fully characterized. We previously developed a renal IRI model in 2/2 2/2

decay-accelerating factor (DAF) and CD59 double-knockout (DAF CD59 ) mice. In this study, we used this model to dissect Downloaded from the pathway(s) by which complement is activated in renal IRI and to evaluate whether C3aR- or C5aR-mediated inflammation or the membrane attack complex was pathogenic. We crossed DAF2/2CD592/2 mice with mice deficient in various complement components or receptors including C3, C4, factor B (fB), factor properdin (fP), mannose-binding lectin, C3aR, C5aR, or Ig and assessed renal IRI in the resulting mutant strains. We found that deletion of C3, fB, fP, C3aR, or C5aR significantly ameliorated renal IRI in DAF2/2CD592/2 mice, whereas deficiency of C4, Ig, or mannose-binding lectin had no effect. Treatment of DAF2/2 2/2

CD59 mice with an anti-C5 mAb reduced renal IRI to a greater degree than did C5aR deﬁciency. We also generated and tested http://www.jimmunol.org/ a function-blocking anti-mouse fP mAb and showed it to ameliorate renal IRI when given to DAF2/2CD592/2 mice 24 h before, but not 4 or 8 h after, ischemia/reperfusion. These results suggest that complement is activated via the alternative pathway during the early phase of reperfusion, and both anaphylatoxin-mediated inﬂammation and the membrane attack complex contribute to tissue injury. Further, they demonstrate a critical role for properdin and support its therapeutic targeting in renal IRI. The Journal of Immunology, 2013, 190: 000–000.

schemia-reperfusion injury (IRI) contributes significantly to binding of these innate immune proteins to the neoantigens acti- morbidity and mortality in various clinical settings, including vates the classical or MBL pathway of complement (3–6). by guest on September 26, 2021 I acute renal failure in allograft and native kidneys (1, 2). The role of complement in renal IRI was also addressed by Animal modeling studies indicated that the complement system multiple investigators using rodent models. Some studies showed plays an important role in the pathogenesis of IRI, but the pathways a critical role for the alternative pathway (AP), while others im- by which complement is activated during ischemia/reperfusion plicated the MBL pathway (7–9), but the mechanistic details of (IR) and the complement effectors that are responsible for tissue complement activation in renal IRI via either pathway remain to injury may be organ specific and remain to be fully characterized. be characterized further. Regarding complement effectors, both Studies using rodent models of skeletal muscle, intestinal, and the membrane attack complex (MAC) and anaphylatoxin receptor heart IRI implicated natural Abs and the mannose-binding lectin (C5aR and C3aR)-mediated signaling on neutrophils and tubular (MBL) pathway of complement in tissue injury (3–6). They led to cells were described to play a pathogenic role in renal IRI (10– the hypothesis that ischemic assaults expose neoantigens on host 15). Additionally, B cell subsets and natural Abs were found to tissues, which are recognized by natural Abs or lectins, and influence renal IRI (16, 17). However, other studies showed that renal IRI is independent of Ig and T lymphocytes (18), and *Department of Pharmacology, Perelman School of Medicine, University of Penn- inhibiting the complement system did not reduce renal IRI, sug- † sylvania, Philadelphia, PA 19104; Institute for Translational Medicine and Thera- gesting a minimal role for complement in the experimental setting peutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and ‡Division of Nephrology and Endocrinology, University of Tokyo School examined (19). of Medicine, Tokyo 113-8655, Japan A challenge in renal IRI studies is to separate complement- Received for publication August 15, 2012. Accepted for publication January 23, mediated injury from that caused by other inflammatory path- 2013. ways that may be triggered, especially when protocols involving This work was supported by National Institutes of Health Grants AI049344, prolonged ischemic periods are used. We previously developed AI085596, AI044970, and GM092108 (to W.-C.S.) and a postdoctoral fellowship from the American Heart Association (to D.G.). a murine model of renal IRI using mice deficient in two mem- Address correspondence and reprint requests to Dr. Wen-Chao Song, Institute for brane complement regulators: decay-accelerating factor (DAF) and Translational Medicine and Therapeutics and Department of Pharmacology, Perel- CD59 (20). By using a protocol of short ischemia (22 min) fol- man School of Medicine, University of Pennsylvania, 1254 BRBII/III, 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address: [email protected] lowed by 24 h of reperfusion, we demonstrated that wild-type (WT) mice sustained only mild renal IRI, whereas DAF2/2 Abbreviations used in this article: AP, alternative pathway; BUN, blood urea nitro- 2/2 gen; CVF, cobra venom factor; DAF, decay-accelerating factor; IR, ischemia/reper- CD59 mice incurred profound renal injury that was comple- fusion; IRI, ischemia-reperfusion injury; MAC, membrane attack complex; MBL, ment dependent, because exacerbation of injury in the double- mannose-binding lectin; PAS, periodic acid-Schiff; WT, wild-type. mutant mice was prevented by complement depletion with cobra Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 venom factor (CVF) (20). In this study, we used this model of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202275 2 PROPERDIN AND RENAL ISCHEMIA-REPERFUSION INJURY heightened complement sensitivity to dissect the activation path- Expression of recombinant mouse properdin way(s) and effector(s) of complement in renal IRI. We found that The mouse properdin full-length cDNA (nt 71–1465, NM_008823.3) was classical and MBL pathways were not involved in this model of amplified by RT-PCR using liver RNA and the following two primers: renal IRI. Rather, complement was activated via the AP in a pro- mprop-S (upstream), 59-CCCGGGGCCACCATGCCTGCTGAAATGCAA- perdin-dependent manner, and both C3aR and C5aR anaphyla- 39 and mprop-AS (downstream), 59-AAGCTTTTAGGGTTTCTTCTCTT- 9 toxin receptors and the MAC contributed to renal IRI. Further, CTGGGTC-3 . This cDNA was then used as a template in subsequent PCR reactions to incorporate an eight-histidine (8xHis) tag and an enterokinase properdin inhibition with a blocking mAb before reperfusion cleavage site at the C terminus of the protein. First, PCR amplification was ameliorated renal IRI, suggesting that antiproperdin therapy may performed using mprop-S (upstream) and R1 (downstream) as primers, 59- have beneficial effects in human IRI. atgatgatgatgatgCTTATCGTCATCGTCGGGTTTCTTCTCTTCTGGGTCT- TTGCAGAC-39 (lower case: 5xHis tag sequence; underlined: enterokinase cleavage site sequence (30); italicized: nucleotide sequence of the 39 coding Materials and Methods region of properdin). The PCR product from the above reaction was used as a template in a further round of PCR using mprop-S (upstream) and R2 Animals (downstream) as primers, 59-CGATCTTTATTAAGATCCACTatgatgatgat- 2 2 2 2 2 2 9 DAF / CD59 / ,fP / , and fPflox/flox-lysozyme-Cre+ mice were gener- gatgatgatgatgCTTATCGTCATCGTC-3 (lower case: 8xHis tag sequence; ated as described previously (20–22). C57BL/6, 129J, and BALB/c WT underlined: enterokinase cleavage site sequence). The final PCR product and MBL-A2/2C2/2 mice (MBL2/2) were purchased from The Jackson was cloned into the pCR2.1 vector and then subcloned at the EcoRI site into Laboratory. The sources of C32/2,C42/2,fB2/2, C3aR2/2, and C5aR2/2 the pCAGGS expression vector (kindly provided by Dr. J. Miyazaki, Osaka mice were described previously (23, 24). Ig2/2 (JHT) mice (25) were University, Osaka, Japan). This construct was transfected into HEK cells kindly provided by Dr. R. Eisenberg (University of Pennsylvania). All 2 2 2 2 mutant mice, with the exception of fB / ,fP / , and fPflox/flox-lysozyme- Downloaded from Cre+ mice, were on the C57BL/6 background. DAF2/2CD592/2 mice were crossed with the relevant mutant mouse strains to generate DAF2/2 CD592/2 C32/2,DAF2/2CD592/2 C42/2,DAF2/2CD592/2 MBL2/2, DAF2/2CD592/2 C3aR2/2,DAF2/2CD592/2 C5aR2/2, and DAF2/2 CD592/2 Ig2/2 mice, for which DAF2/2CD592/2 mice on the C57BL/6 background were used as controls. To generate DAF2/2CD592/2 fB2/2 and DAF2/2CD592/2 fP2/2 mice, DAF2/2CD592/2 mice were crossed http://www.jimmunol.org/ with fB2/2 or fP2/2 mice on a 129/C57BL/6 mixed background. For the study of these mice, littermate DAF2/2CD592/2 mice with the 129/ C57BL/6 background were used as controls. Male mice weighing 25–35 g were used in all renal IRI experiments, and mice aged 7–12 wk were used for serum properdin assays. Mice were housed in a specific pathogen– free facility and were confirmed to be negative for common murine viral pathogens by routine sera analysis. Experiments were conducted by following established guidelines for animal care, and all protocols were ap- proved by the appropriate institutional committees.

Renal IRI induction by guest on September 26, 2021 Renal IR in mice was performed as described previously (20). In short, both renal pedicles were clamped for 22 min using microaneurysm clamps, followed by reperfusion for 24 h. Mice were sacriﬁced, and kidneys were harvested for histologic analysis. Serum samples were collected before ischemia and 24 h after reperfusion. In some experiments, DAF2/2CD592/2 mice were treated i.p. with an anti-C5 mAb (BB5.1) (26) or a control mAb (MOPC-31C; cat. #CCL-130; American Type Culture Collection, Rock- ville, MD) 24 h before ischemia at a dosage of 1 mg/mouse, as previously described (27). In other experiments, mice were treated i.p. with the mouse anti-mouse properdin mAb 14E1 at a dosage of 2 mg/mouse 24 h before ischemia or at 4 or 8 h after reperfusion. Blood urea nitrogen (BUN) levels were measured using urea nitrogen reagents (Sigma-Aldrich, St. Louis, MO) by following the manufacturer’s instructions.

Histopathology and immunohistochemistry Kidneys were fixed in methyl Carnoy’s solution overnight and processed for paraffin embedding. Sections were then stained with periodic acid- Schiff (PAS). Tubular injury was evaluated blindly and scored semiquantitatively, as described previously (20). Cryostat sections (4 mm) of frozen kidneys were stained by immunohistochemistry for neutrophils, comple- 2 2 2 2 FIGURE 1. Deficiency in C3 ameliorates renal IRI in DAF / CD59 / ment factors C3 and C9 (MAC), and properdin, as described previously (20, 28). Briefly, slides were dried and fixed in methanol/acetone. They mice. Renal pedicles were clamped bilaterally for 22 min. Blood was were then treated with a rabbit anti-mouse lactoferrin Ab (for neutrophils; collected before renal pedicle clamping (0 h) and 24 h after reperfusion for 2 2 kindly provided by Dr. C. Teng, National Institute of Environmental Health BUN determinations. (A) Serum levels of BUN in WT (n = 29), DAF / Sciences, Research Triangle Park, NC) or a rabbit anti-rat C9 Ab (for CD592/2 (n = 37), and DAF2/2CD592/2 C32/2 (n = 20) mice. Data MAC; kindly provided by Dr. P. Morgan, University of Wales College of shown are mean 6 SEM. (B) Representative images of PAS, C3, C9, and Medicine, Cardiff, U.K.), which cross-reacts with mouse C9. For staining neutrophil staining in kidney sections of WT, DAF2/2CD592/2, and complement factor C3 and properdin, a biotin-labeled goat anti-mouse C3 DAF2/2CD592/2 C32/2 mice at 24 h after renal IR, showing severe tu- (29) and a rabbit anti-mouse properdin (22) Ab was used, respectively. bular injury (cast formation), deposition of C3 and C9 in peritubular Neutrophils were counted by examining 5–10 fields of view (4003) ran- 2 2 capillaries (arrows), and abundant neutrophil infiltration in DAF / domly selected from the outer medulla and corticomedullary junction on 2/2 2/2 2/2 2/2 each slide. The percentage of tubules with peritubular capillary staining of CD59 mice but not in WT and DAF CD59 C3 mice. Original C3 or C9 in 10 randomly selected fields of view was graded as follows: magnification: PAS, 3200; C3, 3400; C9, 3400; neutrophil, 3200. *p , 2 2 2 2 grade 0, negative; grade 1, ,25%; grade 2, 26–50%; grade 3, 51–75%; 0.0001, versus WT mice, **p , 0.0001, versus DAF / CD59 / mice, grade 4, .75%. Student t test. The Journal of Immunology 3

(American Type Culture Collection) together with a pGK-Hyg vector. After and incubated at 25˚C for 10 min with 100 ml tetramethylbenzidine per- selection on hygromycin for 2 wk, stable HEK cell lines expressing oxidase substrate (BD Pharmingen, San Jose, CA). The reaction was recombinant mouse properdin were established and were confirmed to se- stopped with 50 ml2NH2SO4, and the OD of the samples was measured crete high levels of the protein by Western blot analysis (described below). at 450 nm. Recombinant properdin was purified by Ni2+-chelating chromatography To screen hybridoma cells for desired Abs and to confirm reactivity of (QIAGEN, Valencia, CA) from cell culture supernatants of stable HEK cells purified mAbs, Maxisorp plates (Nunc) were coated for 1 h at 37˚C with (collected 48 h after switching to serum-free DMEM; Life Technologies, 50 ml recombinant properdin (5 mg/ml). Wells were washed three times in Grand Island, NY). washing buffer and then treated with 200 ml 3% BSA/PBS for 1 h at 25˚C. The plates were then incubated with the culture supernatants from hy- SDS-PAGE and Western blot analysis bridoma cells (50 ml/well, diluted at 1:3 or 1:10) or 10-fold serially diluted purified mAbs (50 ml/well, diluted at 10 to 1026 mg/ml) in 3% BSA/PBS The purity of the recombinant mouse properdin was assessed by Coomassie staining of proteins resolved on 8% Tris-glycine gels under reducing for 60 min at 25˚C. Plates were then processed as described above. To measure serum fP levels, Maxisorp plates (Nunc) were coated for 1 h conditions. To screen for stable properdin-expressing HEK cell lines, se- m m rum-free cell culture media were subjected to electrophoresis on 8% Tris- at 37˚C with anti-properdin mAb 14E1 (50 l/well, 2 g/ml). Wells were washed three times in washing buffer (PBS/0.05% Tween 20) and then glycine gels under reducing conditions. Separated proteins on gels were m transferred to polyvinylidene difluoride membranes, which were then treated with 1% BSA/PBS (200 l/well) for 1 h at 25˚C. After adding 50 ml/well of standard (recombinant mouse properdin) or serum samples, both probed with rabbit anti-mouse properdin IgG (2 mg/ml) (22) for 1 h, fol- serially diluted in 1% BSA/PBS, the plates were incubated for 60 min at lowed by HRP-conjugated goat anti-rabbit IgG (1:4000 dilution; Bio-Rad) and the ECL chemiluminescent detection system (Amersham Pharmacia, 25˚C. Wells were washed three times with washing buffer and then incu- Uppsala, Sweden). bated for 60 min at 25˚C after adding biotinylated anti-properdin mAb 5A6-7 (50 ml/well, 2 mg/ml) in 1% BSA/PBS. Biotinylation of mAb 5A6-7 Generation of anti-mouse properdin mAbs was performed using EZ-link Sulfo-NHS-Biotin (Thermo Scientific, Rockford, IL), following the manufacturer’s instructions. Wells were Downloaded from To generate anti-mouse properdin mAbs, a C57BL/6 line of fP-deficient washed three times in washing buffer and incubated for 60 min at 25˚C mice derived from fPflox/flox mice (22) was used. Fully backcrossed (.9 with streptavidin-conjugated HRP (50 ml/well, BD Pharmingen) diluted generations) C57BL/6 fPflox/flox mice were crossed with EIIa-Cre mice, 1:1000. Plates were then processed as described above. which express the Cre recombinase in germline (31). Cre2 progeny with a mutant properdin gene allele (indicative of germline deletion of the fP In vivo inhibition of properdin activity in mice by mAb 14E1 gene) were then intercrossed to generate homozygous properdin-mutant C57BL/6 WT mice weighing 20–22 g were injected i.p. or i.v. with 0.4 or mice. Mice were immunized with 100 mg of recombinant properdin emul- sified in CFA. Boosting immunization (100 mg/mouse each time) was 1.2 mg mAb 14E1. Serum samples were collected at different time points, http://www.jimmunol.org/ performed three times at 2-wk intervals with IFA. Properdin Ab titers in and their LPS-dependent AP complement activity was determined. the immunized mouse sera were determined by ELISA. Prior to hybridoma Assay of serum AP complement activity production, mice were injected i.p. daily with recombinant properdin for three consecutive days (50 mg/mouse in PBS). Splenocytes were harvested Serum AP complement activity was determined as previously described and fused with P3-X63-Ag8.653 myeloma cells (ATCC cat. #CRL-1580). (21). Briefly, ELISA plates were coated with LPS at 4˚C overnight. After After selection and cloning of hybridomas, single clones were grown in washing and blocking with BSA (200 ml/well, 1% in PBS buffer), diluted culture flasks in Hypoxanthine-Thymidine medium (Sigma-Aldrich) con- mouse serum (50 ml/well, 1:10 in Mg2+-EGTA GVB2+ buffer; Sigma- taining Hybridoma-SFM (Life Technologies) and supplemented with FBS Aldrich) was incubated on plates at 37˚C for 1 h. The plates were washed (HyClone), gentamicin (Life Technologies), and penicillin/streptomycin in washing buffer, and plate-bound activated C3 fragments were detected m (Life Technologies). mAbs were purified from serum-free culture super- using HRP-conjugated goat anti-mouse C3 Ab (50 l/well, 1:4000 dilution by guest on September 26, 2021 natant by Protein G affinity chromatography using the A¨ kta FPLC system in 1% PBS buffer; MP Biomedicals, Solon, OH). (Amersham Pharmacia). ELISA Results We previously demonstrated that DAF2/2CD592/2 mice were To measure serum anti-properdin Ab titer in immunized mice, Maxisorp plates (Nunc, Roskilde, Denmark) were coated for 1 h at 37˚C with 50 ml highly susceptible to renal IRI compared with WT controls and recombinant properdin (5 mg/ml). Wells were washed three times in that complement depletion with CVF markedly reduced this sus- washing buffer (PBS/0.05% Tween 20) and then treated with 200 ml1% ceptibility (20). Exacerbated renal IRI in DAF2/2CD592/2 mice BSA/PBS for 1 h at 25˚C. The plates were then incubated with mouse was correlated with microvascular injury and C3/C9 deposition, serum (50 ml/well, diluted at 1:62.5–1:32,000) in 1% BSA/PBS for 60 min at 25˚C. Wells were washed three times with washing buffer and incubated increased neutrophil infiltration into the outer medulla, and higher for 60 min at 25˚C with 50 ml HRP–anti-mouse IgG (Sigma-Aldrich; tubular injury scores (20). To confirm the complement-dependent 1:4000) in 1% BSA/PBS. Wells were washed six times in washing buffer nature of this model as suggested by the CVF experiment, we

Table I. Histological evaluation of kidney sections

Mice Tubular Damage Neutrophil Infiltration C3 Staining C9 Staining WT 0.3 6 0.4* 16.0 6 8.8* 0.3 6 0.3* 0.3 6 0.2* DAF2/2CD592/2 3.2 6 0.6 96.5 6 13.4 1.4 6 0.2 1.3 6 0.2 C32/2DAF2/2CD592/2 0.4 6 0.4* 33.2 6 11.0* 0 6 0* 0.4 6 0.2* Ig2/2DAF2/2CD592/2 3.2 6 0.7 95.7 6 18.9 1.3 6 0.5 1.3 6 0.2 C42/2DAF2/2CD592/2 3.6 6 0.5 90.7 6 20.2 1.3 6 0.3 1.1 6 0.3 MBL2/2DAF2/2CD592/2 3.1 6 1.0 95.0 6 15.9 1.1 6 0.1 1.1 6 0.1 fB+/+DAF2/2CD592/2 3.3 6 0.7 96.8 6 15.3 1.2 6 0.2 1.3 6 0.2 fB2/2DAF2/2CD592/2 0.5 6 0.6** 19.2 6 4.9** 0.1 6 0.1** 0.4 6 0.2** fP+/+DAF2/2CD592/2 2.6 6 0.9 101.0 6 15.3 1.0 6 0.2 1.3 6 0.3 fP2/2DAF2/2CD592/2 1.1 6 1.2*** 58.0 6 11.6*** 0.4 6 0.1*** 0.6 6 0.2*** C5aR2/2DAF2/2CD592/2 1.9 6 1.0* 46.1 6 10.9* 0.6 6 0.4* 0.7 6 0.2* C3aR2/2DAF2/2CD592/2 0.9 6 1.0* 49.7 6 11.9* 0.3 6 0.3* 0.7 6 0.2* Seven mice from each group were randomly selected for histological measures of tubular damage, neutrophil infiltration, and deposition of C3 and C9 in peritubular capillaries. Neutrophil values are numbers per field of view. Tubular damage and C3 and C9 staining were scored semiquantitatively, as described in the Materials and Methods. Data are mean 6 SEM. *p , 0.05, versus DAF2/2CD592/2 mice, **p , 0.05, versus fB+/+DAF2/2CD592/2 mice, ***p , 0.05, versus fP+/+ DAF2/2CD592/2 mice. 4 PROPERDIN AND RENAL ISCHEMIA-REPERFUSION INJURY crossed DAF2/2CD592/2 mice with C32/2 mice to generate DAF2/2CD592/2 C32/2 mice and assessed renal IRI. As we observed before, 22 min of bilateral renal ischemia followed by 24 h of reperfusion led to more severe renal IRI in DAF2/2CD592/2 mice than in WT mice (Fig. 1, Table I). In WT mice, the BUN level increased from 24.2 6 0.2 mg/dl before IR to 68.8 6 8.8 mg/ dl after IR, whereas it increased from 24.4 6 0.5 to 135.6 6 8.4 mg/dl in DAF2/2CD592/2 mice (Fig. 1A). Importantly, we found that C3 deficiency in DAF2/2CD592/2 mice reduced BUN to WT mouse levels (56 6 7.5 mg/dl) (Fig. 1A). Corroborating the BUN data, renal histology revealed significantly milder tubular injury, lower numbers of neutrophils in the outer medulla, and less C3 and C9 deposition in peritubular capillaries of DAF2/2CD592/2 C32/2 mice compared with DAF2/2CD592/2 mice (Fig. 1B, Table I). Thus, exacerbation of renal IRI in DAF2/2CD592/2 mice was confirmed to be critically dependent on C3. Natural Abs and the lectin pathway of complement have been implicated in other rodent models of IRI (3–6). To determine 2 2 whether they might also be involved in renal IRI of DAF / Downloaded from CD592/2 mice, we crossed DAF2/2CD592/2 mice with Ig2/2, C42/2, or MBL-A/MBL-C double-knockout (MBL2/2) mice. We found that deficiency of Abs, C4, or MBL did not significantly affect the susceptibility of DAF2/2CD592/2 mice to renal IRI (Fig. 2, Table I). Thus, after renal IR challenge, DAF2/2CD592/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 Ig ,DAF CD59 C4 , and DAF CD59 MBL http://www.jimmunol.org/ mice had BUN levels that were not significantly different from each other or from DAF2/2CD592/2 mice (Fig. 2A). This result was further corroborated by histological evaluations of tubular injury, neutrophil infiltration, and complement staining in peritubular capillaries among the different groups (Fig. 2B, Table I). Together, the data suggested that, unlike skeletal muscle, intestinal, and myocardial IRI models (3–6), complement-dependent renal IRI in DAF2/2CD592/2 mice was not mediated by natural Abs and neither the classical nor the MBL pathway was involved. by guest on September 26, 2021 In light of the above findings, we next examined the role of the 2/2 2/2 AP of complement in this process by crossing DAF CD59 FIGURE 2. Deficiency in Abs, C4, or MBL does not affect renal IRI in mice with mice deficient in complement fB. Examination of DAF2/2CD592/2 mice. Blood was collected before renal pedicle clamp- 2/2 2/2 2/2 DAF CD59 fB mice showed that renal IRI was mark- ing (0 h) and 24 h after reperfusion for BUN determinations. (A) Serum edly reduced, with BUN and tubular injury scores essentially levels of BUN in DAF2/2CD592/2 (n = 37), DAF2/2CD592/2 Ig2/2 (n = reverting to WT mouse levels (Figs. 1, 3, Table I). This result 25), DAF2/2CD592/2 C42/2 (n = 37), and DAF2/2CD592/2 MBL2/2 indicated that exacerbated renal IRI in DAF2/2CD592/2 mice (n = 15) mice. (B) Representative images of PAS, C3, C9, and neutrophil 2/2 2/2 2/2 2/2 2/2 was mediated by the AP complement. To further dissect AP staining in kidney sections of DAF CD59 Ig ,DAF CD59 2/2 2/2 2/2 2/2 complement activation under this setting, we crossed DAF2/2 C4 , and DAF CD59 MBL mice at 24 h after renal IR, CD592/2 mice with mice deficient in fP (fP2/2). Properdin is the showing no effect on tubular injury, deposition of C3 and C9 in peritubular capillaries (arrows), and neutrophil infiltration by Ig, C4, or MBL defi- only known positive regulator of complement, and it promotes AP ciency. Original magnification: PAS, 3200; C3, 3400; C9, 3400; neu- complement activation by stabilizing the C3 convertase C3bBb trophil, 3200. BUN data for DAF2/2CD592/2 mice in (A) are the same as (32, 33). Recent evidence suggested that fP may also bind to in Fig. 1 and are presented for comparison. susceptible surfaces to initiate AP complement activation (34, 35). Comparison of DAF2/2CD592/2 fP2/2 mice and their fP-sufficient littermates revealed that fP played a major role in renal IRI, be- C5aR-mediated inflammatory response was involved. We found cause BUN, tubular injury, neutrophil infiltration, and microvas- that, compared with DAF2/2CD592/2 mice, renal IRI in DAF2/2 cular complement deposition in DAF2/2CD592/2 fP2/2 mice CD592/2 C3aR2/2 and DAF2/2CD592/2 C5aR2/2 mice was were significantly reduced (Fig. 3A, 3B, Table I). Notably, we significantly ameliorated, as assessed by BUN and histological detected fP staining in peritubular capillaries of IRI-challenged parameters (Fig. 4A, Table I). However, the magnitude of BUN DAF2/2CD592/2 fP+/+ mice but not in DAF2/2CD592/2 fP2/2 reduction by either C3aR or C5aR deficiency was less than when mice littermates (Fig. 3C). the C3 or fB gene was deleted (Figs. 1, 3), suggesting that both Activation of the AP complement generates as effectors the C3aR- and C5aR-mediated inflammation contributed to renal IRI, anaphylatoxins C3a and C5a, as well as the MAC. The anaphy- and deletion of either effector pathway was not sufficient to latoxins function through binding to their cognate receptors, C3aR completely rescue the DAF2/2CD592/2 mouse phenotype. To and C5aR on neutrophils and macrophages, and play a major role further investigate whether MAC-mediated cellular injury also in complement-mediated inflammatory injury (36). To characterize played a role in the pathogenesis of renal IRI, we used an anti-C5 the complement effectors responsible for exacerbated renal IRI in mAb that blocks C5 cleavage and prevents the generation of C5a, DAF2/2CD592/2 mice, we crossed DAF2/2CD592/2 mice with as well as the MAC (26). This experiment demonstrated that C3aR2/2 and C5aR2/2 mice to determine whether a C3aR- or blocking C5 in DAF2/2CD592/2 mice was more effective than The Journal of Immunology 5

A C DAF-/-CD59-/-fP+/+

** BUN (mg/dl) * DAF-/-CD59-/-fP-/-

0 24 0 24 0 24 0 24 hour

DAF-/-CD59-/-fB+/+ DAF-/-CD59-/-fP+/+ DAF-/-CD59-/-fB-/- DAF-/-CD59-/-fP-/-

PAS Downloaded from C3

C9 http://www.jimmunol.org/

Neutrophil FIGURE 4. Both anaphylatoxin receptors C3aR and C5aR and the MAC contribute to renal IRI in DAF2/2CD592/2 mice. Blood was collected before renal pedicle clamping (0 h) and 24 h after reperfusion for BUN -/- -/- +/+ -/- -/- -/- -/- -/- +/+ -/- -/- -/- DAF CD59 fB DAF CD59 fB DAF CD59 fP DAF CD59 fP determinations. (A) Serum levels of BUN at 24 h after renal IR in DAF2/2 2/2 FIGURE 3. Deficiency in fB or fP ameliorates renal IRI in DAF CD592/2 C3aR2/2 (n = 20) and DAF2/2CD592/2 C5aR2/2 mice (n = 33) 2/2 CD59 mice. Blood was collected before renal pedicle clamping (0 h) were significantly lower than in DAF2/2CD592/2 mice (n = 37). DAF2/2 and 24 h after reperfusion for BUN determinations. (A) Serum levels of CD592/2 mouse data are the same as in Fig. 1 and are presented for com- 2/2 2/2 2/2 2/2 2/2 2/2 BUN in DAF CD59 fB (n = 9), DAF CD59 fP (n = parison. Data shown are mean 6 SEM. *p , 0.001, **p , 0.01, versus 14), and fB- or fP-sufficient littermate controls (n = 10 and n = 11, re- DAF2/2CD592/2 mice, Student t test. (B) Serum levels of BUN at 24 h spectively). Data shown are mean 6 SEM. (B) Representative images of after renal IR in DAF2/2CD592/2 mice treated with an anti-C5 mAb by guest on September 26, 2021 2/2 PAS, C3, C9, and neutrophil staining in kidney sections of DAF (BB5.1, n = 13) or a control mAb (MOPC, n = 7) 24 h before ischemia 2/2 +/+ 2/2 2/2 2/2 2/2 2/2 +/+ CD59 fB ,DAF CD59 fB ,DAF CD59 fP , and surgery. Data shown are mean 6 SEM. *p , 0.001, versus control IgG, 2/2 2/2 2/2 DAF CD59 fP mice at 24 h after renal IR, showing a significant Student t test. reduction of tubular injury, deposition of C3 and C9 in peritubular capillaries (arrows), and neutrophil infiltration in DAF2/2CD592/2 fB2/2 and DAF2/2CD592/2 fP2/2 mice. (C) Representative images of properdin 2 2 2 2 2 2 2 2 example, injection of 0.4 and 1.2 mg of 14E1 to WT mice blocked staining in kidney sections of DAF / CD59 / fP+/+ and DAF / CD59 / fP2/2 mice at 24 h after renal IR, showing deposition of properdin in LPS-dependent AP complement activity for 2 and 9 d, respec- peritubular capillaries in DAF2/2CD592/2 fP+/+ mice but not in DAF2/2 tively (Fig. 5D). By sandwich ELISA assays using these mAbs, CD592/2 fP2/2 mice. Original magnification: PAS, 3200; C3, 3400; we surveyed serum fP levels in WT mice of different genetic C9, 3400; neutrophil, 3200; properdin, 3400. Data shown are mean 6 background and in mutant mice lacking various complement SEM. *p , 0.0001 versus DAF2/2CD592/2 fB+/+ mice, **p , 0.001 components, as well as in fPflox/flox–lysozyme–Cre+ mice with 2 2 2 2 versus DAF / CD59 / fP+/+ mice. Student t test. tissue-specific fP gene deletion in myeloid lineage cells (22). We found that serum fP levels in C57BL/6J, 129J, and BALB/c mice ranged between 15 and 20 mg/ml and that serum levels was C5aR gene deletion in reducing renal IRI (Fig. 4B, BUN for of fPflox/flox–lysozyme–Cre+ mice were ,5% WT mouse levels DAF2/2CD592/2 C5aR2/2 mice, 85.2 6 10.2 mg/dl; BUN for (Fig. 5E), suggesting that myeloid lineage cells are the major DAF2/2CD592/2 mice treated with anti-C5 mAb, 57.1 6 12.1 source of serum fP. Of interest, we found serum properdin levels in mg/dl), suggesting that the MAC was also likely to have con- C32/2,fB2/2,andC42/2 mice to be significantly lower than in tributed to renal IRI. WT mice (Fig. 5F). The finding that properdin gene deletion ameliorated renal IRI in We next tested whether administration of a function-blocking DAF2/2CD592/2 mice prompted us to investigate whether pro- mAb against fP to DAF2/2CD592/2 mice before or after re- perdin might be amenable to therapeutic targeting in the setting of nal IR challenge would be effective at preventing renal injury. renal IRI. To test this concept, we expressed and purified DAF2/2CD592/2 mice were treated by i.p. injection of mAb recombinant mouse fP in HEK293 cells (Fig. 5A) and used it to 14E1 at 24 h before ischemia or at 4 or 8 h after reperfusion. As immunize a line of fP2/2 mice obtained by germline deletion of shown in Fig. 6, we found that systemic blockade of fP before the floxed fP gene (22). By this approach, we produced several ischemia significantly ameliorated renal IRI, and this treatment high-affinity mouse anti-mouse fP mAbs, one of which (14E1) appeared to be as effective as fP gene deletion (Fig. 3). In contrast, was found to be function blocking in an LPS-dependent AP blocking fP at 4 h after reperfusion was marginally effective, and complement-activation assay (Fig. 5B, 5C). When tested in mice, the protection was completely lost if the mAb was given at 8 h 14E1 effectively blocked AP complement activity in vivo. For postreperfusion (Fig. 6). These results suggested that fP played 6 PROPERDIN AND RENAL ISCHEMIA-REPERFUSION INJURY Downloaded from http://www.jimmunol.org/

FIGURE 5. Generation and characterization of mAbs against mouse properdin. (A) SDS-PAGE analysis of purified recombinant mouse properdin. (B) by guest on September 26, 2021 Reactivity of mAbs with mouse serum properdin by sandwich ELISA. Two mAbs, 14E1 and 5A6-7, were used as the capture and detection Ab, respectively, in the sandwich ELISA assay. Properdin was detected in WT mouse sera (n = 2) but not in the sera of a line of fP knockout mice (n = 2) derived from fPflox/flox mice after Cre-mediated germline deletion (fPKO). (C) At 2.5 mg/ml or above, mAb 14E1 blocked mouse properdin activity in an LPS- induced AP complement activation assay using WT mouse serum (1:10 dilution). fP2/2 mouse serum of the same dilution and without 14E1 mAb added (3) was used as a control. (D) In vivo effect of mAb 14E1. WT mice (n = 3/group) were given a single i.p. injection of 0.4 mg (N) or 1.2 mg (;) of the Ab. Serum samples were collected at the indicated time points and assayed (at 1:10 dilution) for LPS-induced AP complement activity as in (C). Similarly diluted fP2/2 mouse serum (d) was used as a control. (E) Serum levels of properdin in WT mice of different genetic backgrounds and in mice with myeloid lineage cell–specific deletion of properdin (LysCre+-fPflox/flox) and their Cre2 littermates. Sera from fPKO mice were used as a negative control. C57BL/6 (n = 5), 129J (n = 4), BALB/c (n = 4), fPKO (n = 4), LysCre+-fPflox/flox (n = 6), and LysCre2-fPflox/flox (n = 5). Values are mean 6 SEM. *p , 0.0001, versus C57BL/6, **p , 0.001, versus LysCre2-fPflox/flox, Student t test. (F) Serum properdin levels in C57BL/6 [n = 5, same as in (E)], C32/2 (n = 6), fB2/2 (n = 4), C42/2 (n = 5), and DAF2/2CD592/2 (n = 5) mice, showing that the level was significantly lower in C32/2,fB2/2, and C42/2 mice but not in DAF2/2 CD592/2 mice. Values are mean 6 SEM. *p , 0.01, **p , 0.05, versus WT. Student t test. Mr, Molecular mass markers.

a major role in AP complement activation in the initial phase of 37). Other investigators reported natural Abs to be pathogenic in reperfusion, and therapeutic targeting of circulating properdin to intestinal IRI by recognizing annexin IV as a neoantigen on is- reduce renal IRI is feasible and effective when carried out pro- chemic tissues (38). In addition, deficiency of MBL or mannose- phylactically (e.g., in the setting of renal transplantation). binding lectin-associated serine protease 2 was found to be protective in mouse and rat models of myocardial, intestinal, and Discussion renal IRI (6, 9, 39, 40). In the current study, we confirmed that In this study, we used the DAF2/2CD592/2 mouse model to dis- exacerbation of renal IRI in DAF2/2CD592/2 mice was com- sect the pathways and effectors of complement activation in renal plement dependent, because C3 deficiency rescued the phenotype. IRI. In contrast to several other rodent IRI models in which However, we found that deficiency of Ig, C4, or MBL had no natural Abs and the classical or MBL pathway were implicated effect on renal IRI. Thus, our data exclude any involvement of the (3–6), we found that complement was activated via the AP in renal classical pathway or MBL in the complement-mediated renal IRI IRI of DAF2/2CD592/2 mice and that properdin played a major of DAF2/2CD592/2 mice. It is unknown why MBL deficiency pathogenic role in this process. In previous studies of murine ameliorated renal IRI in other studies (9) but not in the DAF2/2 models of intestinal and skeletal muscle IRI, nonmuscle myosin H CD592/2 mouse model in this study. It is possible that the dis- chain II was identified as a self-Ag that became exposed on host crepancy is related to differences in the experimental protocols tissues after ischemia, and binding of this protein by a natural IgM used (e.g., WT versus DAF2/2CD592/2 mice, length of ische- Ab (CM-22) triggered MBL pathway complement activation (4, mia). However, it should be noted that, although MBL and C4 are The Journal of Immunology 7

a recent study using mice deficient in C3aR and/or C5aR also showed that both C3aR- and C5aR-mediated pathways contributed to renal IRI (12). Because C5 activation generates C5a, as well as C5b, which initiates the formation of the lytic MAC on target cells, we also evaluated the involvement of the MAC in renal IRI of DAF2/2CD592/2 mice using a C5-blocking mAb. We found that administration of anti-C5 mAb to DAF2/2CD592/2 mice reduced renal IRI to a greater degree than did C5aR gene deletion, implying that the MAC had also contributed to renal IRI. This conclusion is also supported by our earlier observation that renal IRI was more severe in DAF2/2CD592/2 mice than in DAF2/2 mice (20), presumably reflecting increased MAC formation in the absence of CD59 as a MAC inhibitor. As the only known positive regulator of the complement system, properdin facilitates AP complement activation either by stabilizing the C3 convertase C3bBb or by binding to susceptible surfaces to serve as a platform for de novo C3bBb assembly (32, 33, FIGURE 6. Systemic inhibition of properdin with mAb before, but not 35). Given that genetic deficiency of properdin was protective and after, IR ameliorates renal IRI in DAF2/2CD592/2 mice. Mice were that both C3 and terminal complement activation effectors were Downloaded from treated with a control IgG (MOPC, n = 10) at 24 h before renal IR surgery pathogenic in renal IRI, we tested the concept of therapeutic in- or with a mouse anti-mouse properdin mAb (14E1) at 24 h before IR hibition of properdin in renal IRI. We successfully generated surgery (n = 7) and at 4 h (n = 4) and 8 h (n = 5) after reperfusion. Blood function-blocking mAbs against mouse properdin by immunizing was collected before renal pedicle clamping (0 h) and 24 h after reper- an fP-deficient mouse with recombinant mouse fP protein. Using fusion for BUN determinations. Data shown are mean 6 SEM. *p , 0.05, a newly developed ELISA assay, we showed that systemic pro- versus control IgG, Student t test. perdin is mainly produced by myeloid lineage cells, and its serum http://www.jimmunol.org/ levels ranged between 15 and 20 mg/ml in WT mice of different not involved in the renal IRI of DAF2/2CD592/2 mice, we cannot genetic backgrounds. Interestingly, serum properdin levels in rule out the possibility that a “bypass” lectin pathway (i.e., direct C32/2,fB2/2, and C42/2 mice were significantly lower than that C3 cleavage) (40, 41) mediated by ficolins may have played a role. of WT mice, implying that either the number of myeloid lineage Such a scenario could be tested in future experiments by blocking cells or their properdin-secreting activity was affected in these the function of mannose-binding lectin-associated serine protease mutant mice. We found that systemic inhibition of properdin by 2, the key activation enzyme of the lectin pathway, in DAF2/2 mAb before reperfusion significantly reduced renal IRI in DAF2/2 CD592/2 mice. CD592/2 mice. The effect of mAb inhibition on reducing renal In contrast to the noninvolvement of Abs, MBL, and the classical IRI was similar to that of properdin gene deletion, suggesting that by guest on September 26, 2021 pathway, we found that renal IRI in DAF2/2CD592/2 mice was the pathogenic role of properdin in this disease setting is fully completely dependent on the AP, because fB deficiency prevented amenable to pharmacological targeting. By manipulating the exacerbation of renal IRI. Furthermore, we showed that properdin, timing of properdin inhibition, we further showed that the path- a positive regulator of the AP, played a major pathogenic role in ogenic role of properdin and, therefore, the AP complement in this process. Whether the AP was the primary and sole pathway renal IRI, is imparted during the initial phase (,4 h) of reperfu- activated or functioned as an amplification loop secondary to other sion after ischemia. triggers, such as the ficolins, remains to be determined. It is cer- In summary, by using DAF2/2CD592/2 mice with an increased tainly possible that the AP was activated independently. Ischemic sensitivity to complement injury, we dissected the complement challenge may impair the function of the remaining membrane activation pathways and pathogenic effectors in renal IRI. Our complement regulator Crry in DAF2/2CD592/2 mice (42), and genetic and pharmacological data identify the AP, properdin, ischemia-induced plasma membrane remodeling on endothelial C3aR, C5aR, and the MAC as components responsible for renal cells may reduce their affinity for the fluid-phase complement IRI and exclude the participation of natural Abs, C4, and MBL. regulator factor H (43). Together, these events would render the The finding of a beneficial effect of properdin targeting in renal ischemic tissues susceptible to spontaneous AP complement at- IRI adds to a growing list of AP complement–mediated patholo- tack, which was likely to be facilitated by properdin as a positive gies for which antiproperdin therapy may be effective (22, 44), regulator. although recent studies also showed settings in which properdin Data from the literature suggest that C3a–C3aR interaction may inhibition may not be desirable (28, 45). play a role in renal IRI. It was shown that bilateral renal IR in mice resulted in a significant increase in systemic levels of C3a, and C3a was required for the induction of MIP-2 and KC, potent Acknowledgments neutrophil chemotaxins, after renal IR (11). These observations We thank Dr. Christina Teng for anti-lactoferrin Abs, Dr. Paul Morgan for anti-C9 Abs, Dr. Robert Eisenberg for Ig2/2 mice, and Dr. J. Miyazaki for are in agreement with our finding that C3aR deficiency was par- the pCAGGS expression vector. tially protective for renal IRI in DAF2/2CD592/2 mice. Likewise, we found that C5aR deficiency also ameliorated renal IRI, suggesting that C5a may have contributed to neutrophil infiltration Disclosures into the outer medulla of the kidney, as we described previously The authors have no financial conflicts of interest. (20). That C5a-mediated inflammation contributed to renal IRI in 2/2 2/2 DAF CD59 mice is also consistent with findings from other References renal IRI studies in which C5aR function was inhibited by a small 1. Thadhani, R., M. Pascual, and J. V. Bonventre. 1996. Acute renal failure. N. molecule antagonist or small interfering RNA (13, 14). Of interest, Engl. J. Med. 334: 1448–1460. 8 PROPERDIN AND RENAL ISCHEMIA-REPERFUSION INJURY

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