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W F1 mice and alternative pathway inhibition
Molecular Immunology 49 (2011) 317–323
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The dual role of complement in the progression of renal disease in NZB/W F1 mice and alternative pathway inhibition Hideharu Sekine a , Phillip Ruiz b , Gary S. Gilkeson a , Stephen Tomlinson c,∗ a b c
Department of Medicine, Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston, SC 29425, USA Department of Pathology, University of Miami, School of Medicine, Miami, FL 33136, USA Department of Microbiology and Immunology, Darby Children’s Research Institute, Medical University of South Carolina, Charleston, SC 29425, USA
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Article history: Received 28 March 2011 Received in revised form 1 September 2011 Accepted 5 September 2011 Available online 14 October 2011 Keywords: Complement Lupus Factor H Kidney Autoimmunity
a b s t r a c t Complement plays a dual role in the progression of systemic lupus erythematosus since it has important protective functions, such as the clearance of immune complexes and apoptotic cells, but is also a mediator of renal inflammation. To investigate this balance in a clinically relevant setting, we investigated how targeted inhibition of all complement pathways vs. targeted inhibition of only the alternative pathway impacts immune and therapeutic outcomes in NZB/W F1 mice. Following onset of proteinuria, mice were injected twice weekly with CR2-fH (inhibits alternative pathway), CR2-Crry (inhibits all pathways at C3 activation step), sCR2 (C3d targeting vehicle) or saline. Sera were analyzed every 2 weeks for anti-dsDNA antibody levels, and urinary albumin excretion was determined. Kidneys were collected for histological evaluation at 32 weeks. Compared to the control group, all CR2-fH, CR2-Crry and sCR2 treated groups showed significantly reduced serum anti-dsDNA antibody levels and strong trends towards reduced glomerular IgG deposition levels. Glomerular C3 deposition levels were also significantly reduced in all three-treated groups. However, significant reductions of disease activity (albuminuria and glomerulonephritis) were only seen in the CR2-fH treated group. These data highlight the dual role played by complement in the pathogenesis of lupus, and demonstrate a benefit of selectively inhibiting the alternative complement pathway, presumably because of protective contributions from the classical and/or lectin pathways. The sCR2 targeting moiety appears to be contributing to therapeutic outcome via modulation of autoimmunity. Furthermore, these results are largely consistent with our previous data using the MRL/lpr lupus model, thus broadening the significance of these findings. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction The complement system plays an important role in host defense and immune regulation, but also plays a pathogenic role in many inflammatory and autoimmune diseases, including systemic lupus erythematosus (SLE or lupus) (Bao and Quigg, 2007; Chen et al., 2010). SLE is a prototypic autoimmune disease characterized by production of autoantibodies directed against a broad range of selfantigens, with formation and/or deposition of immune complexes (ICs) in targeted organs, followed by activation of the complement cascade leading to a local inflammatory process. The kidney is a major site of IC formation/deposition in lupus, and renal inflammation (i.e. lupus nephritis) is a major cause of morbidity and
Abbreviations: CR, complement receptor; SCR, short consensus repeat; SLE, systemic lupus erythematosus. ∗ Corresponding author. Tel.: +1 843 792 1450; fax: +1 843 792 2462. E-mail address: [email protected] (S. Tomlinson). 0161-5890/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2011.09.015
mortality, both in human SLE and in murine models of the disease. IC formation/deposition in glomeruli triggers activation of the classical pathway upon binding of C1q to an exposed antibody Fc region. C1r and C1s serine proteases, that are associated with C1q, are then activated and split C2 and C4 to form the enzyme complex C3 convertase, followed by C3 activation resulting in activation of the downstream anaphylotoxins (C3a and C5a), formation of the membrane attack complex (MAC, C5b–C9) and activation of the alternative complement pathway amplification loop through C3b. Paradoxically, patients with homozygous deficiencies of early components of the classical pathway (C1q, C1r/s or C4), have an increased prevalence of lupus and lupus-like disease (>80%) (Bao and Quigg, 2007; Walport, 2001). Homozygous deficiencies of C3, a protein that plays a pivotal role in all three complement activation pathways, is associated with membranoproliferative glomerulonephritis (Botto and Walport, 1993), but only rarely lupus (Pickering et al., 2000). This apparent contrasting role of complement in lupus is denoted “the lupus paradox”, and is explained by the fact that complement also plays a role in the clearance
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of apoptotic cells that are a potential source of self-antigens. In fact, C1q deficient mice have increased mortality and high titers of autoantibodies, with 25% of C1q−/− mice developing glomerulonephritis with glomerular IC deposits and multiple apoptotic bodies (Botto et al., 1998). C1q and C4 deficiency are also associated with the development/acceleration of lupus-like disease in mice on an autoimmune background (Einav et al., 2002; Mitchell et al., 2002), and C1q−/− and C4−/− mice (on 129/Sv × C57BL/6 background) exhibit an impaired ability to clear apoptotic cell bodies. Thus, these data support the hypothesis that the classical pathway plays a protective role in the development of lupus via its role in the clearance of apoptotic cells that otherwise provide a source of autoantigens to fuel autoimmune disease (Manderson et al., 2004). The alternative pathway is thought to play a key role in the inflammatory process in lupus nephritis. Indeed, previous studies have demonstrated a significant role for the alternative pathway in the development of renal disease in MRL/lpr mice, a murine strain that spontaneously develops lupus-like disease, including glomerulonephritis. For example, MRL/lpr mice deficient in either of the alternative pathway proteins factor B (fB) or factor D (fD) were protected from renal disease (Elliott et al., 2004; Watanabe et al., 2000). On the other hand, C3−/− MRL/lpr mice showed no improvement in pathologic renal disease and even worse albuminuria and increased glomerular IC deposits, likely due to decreased IC clearance by C3b (Sekine et al., 2001). Other complement inhibitors also showed protective effects for renal disease in MRL/lpr mice. The rodent membrane complement inhibitor Crry (complement receptor-related gene y) inhibits all complement pathways at the level of C3 by inhibiting C3 convertases, and administration of recombinant soluble forms of Crry (Crry-Ig (Quigg et al., 1998) and CR2-Crry (Atkinson et al., 2008)) provided protection against pathologic renal injury in MRL/lpr mice. However, treatment with the systemic inhibitor Crry-Ig resulted in increased levels of circulating ICs and no reduction in serum autoantibody levels compared to untreated controls. In contrast, treatment with CR2-Crry, which targets Crry to C3 breakdown products deposited at sites of complement activation, resulted in reduced serum autoantibody levels and no increase in circulating ICs. These different outcomes may be related to the systemic vs. localized nature of complement inhibition by CrryIg vs. CR2-Crry, respectively. Interestingly, the effect of CR2-Crry treatment on serum autoantibody levels, glomerular IC deposits and glomerulonephritis in MRL/lpr mice was observed only in mice receiving once a week administration (Atkinson et al., 2008), while mice receiving twice a week dosing did not demonstrate benefit in lupus like disease, possibly due to more complete and long lasting inhibition of complement. Based on these data, we recently investigated the effect of a new targeted inhibitor specific for the alternative pathway, CR2-fH, on renal disease in MRL/lpr mice. Compared to saline-injected controls, mice treated with CR2-fH had decreased serum autoantibodies, circulating IC, glomerular IC, C3 deposits, albuminuria, renal scores (pathological assessment of glomerulonephritis), vasculitis, and survival (Sekine et al., 2011). Collectively, the above studies indicate that targeted and selective inhibition of the alternative complement pathway is an effective treatment for lupus-like disease in MRL/lpr mice, and is more effective than inhibition of all complement pathways at the C3 level. However, there is a significant concern about different treatment outcomes in the different murine models of lupus given differences in pathogenesis between strains. Indeed, Fc␥RI and Fc␥RIII-deficient NZB/W F1 mice, another widely studied lupus-prone murine strain, had significantly decreased renal injury compared to littermate controls (Clynes et al., 1998), while MRL/lpr mice deficient in these same Fc receptors, still developed severe renal disease (Matsumoto et al., 2003). Interferon
(IFN)-␣, a type I IFN, is implicated in the pathogenesis of human SLE. However, it plays opposite roles in the development of lupus between murine lupus models. Treatment with IFN-␣ induces early lethal lupus nephritis in pre-autoimmune NZB/W F1 mice (Mathian et al., 2005), and type-1 IFN receptor deficiency reduces lupus-like disease (Santiago-Raber et al., 2003), indicating a pathogenic role for IFN-␣ in NZB/W F1 mice. In contrast, IFN-RI-deficient MRL/lpr mice developed worsened lupus-like disease, indicating a protective role of IFN-␣ in MRL/lpr (Hron and Peng, 2004). Thus, as in human lupus, efficacy of a therapy in one strain or patient, does not necessarily translate into effectiveness in all strains or patients. To determine whether selective alternative pathway inhibition is an effective treatment for lupus-like renal disease in a different lupus prone strain with different pathogenic mechanisms of disease (and thus also broaden potential clinical significance), we investigated and compared the effects of CR2-fH, CR2-Crry and sCR2 on lupus-like renal disease in NZB/W F1 female mice. 2. Materials and methods 2.1. Preparation and purification of CR2-fH, CR2-Crry and sCR2 The recombinant proteins CR2-fH, CR2-Crry and soluble CR2 (sCR2) were produced and purified as described previously (Atkinson et al., 2005; Huang et al., 2008). Proteins were expressed in Chinese hamster ovary (CHO) cells transfected with plasmids encoding either mouse CR2-fH, CR2-Crry or CR2, and purified from culture supernatants by anti-mouse CR2 (mAb 7G6) affinity chromatography as described (Atkinson et al., 2005; Huang et al., 2008). 2.2. Mice Female NZB/W F1 mice (#100008) were purchased from The Jackson Laboratory (Bar Harbor, ME). After the onset of renal disease at week 23 as determined by urinary protein excretion (>0.1 mg/mouse/day), the NZB/W F1 mice were randomized into four groups for biweekly treatment from weeks 23 to 31 as follows: (i) CR2-fH treatment group (n = 10, 0.4 mg twice a week), (ii) CR2-Crry treatment group (n = 10, 0.25 mg twice a week), (iii) sCR2 treatment group (n = 10, 0.18 mg twice a week) and (iv) control (saline) treatment group (n = 14, twice a week). CR2-fH, CR2-Crry, sCR2 and saline were administered by intraperitoneal (i.p.) injection. The dose and dosing frequency were selected to investigate the effect and potential relative benefits of targeted complement inhibition and were based on prior pharmacokinetic and efficacy experiments (Atkinson et al., 2005). All work with mice was approved by the Medical University of South Carolina Animal Protocols Review Board and was performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. 2.3. ELISA Measurements of serum anti-dsDNA antibody levels were determined by ELISA as previously described (Sekine et al., 2006). Pooled serum samples from 23-week-old MRL/lpr and C57BL/6 (B6) mice were used as positive and negative controls, respectively. 2.4. Urine albumin excretion Mice were placed in metabolic cages for 24 h urine collection every 2 weeks beginning at week 23. To prevent bacterial growth, antibiotics (ampicillin, gentamicin and chloramphenicol) were added to collection tubes. Urinary albumin excretion was determined by ELISA using a standard curve of known concentrations of mouse albumin (Bethyl Laboratories, Montgomery, TX) as
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previously described (Sekine et al., 2006). Results are expressed as milligrams of albumin per mouse per day. 2.5. Pathology At the time of sacrifice (32 weeks), the kidneys were removed. One kidney was snap frozen in liquid nitrogen and placed in OCT medium. 4-mm thick frozen sections were stained with fluorescein-conjugated anti-mouse IgG (MP Biomedical, Solon, OH) or anti-mouse C3 (MP Biomedical). Immunofluorescence slides were read blinded and graded on a scale of 0–3+ for fluorescence intensity (0, none; 1+, mild staining; 2+, moderate staining; 3+, high staining). The other kidney was fixed in 10% buffered formalin, embedded in paraffin, and sectioned before staining with hematoxylin and eosin (H&E). Slides were examined in a blinded fashion and graded for glomerular inflammation, proliferation, crescent formation and necrosis. Interstitial and tubular changes were also noted. Scores from 0 to 4+ (0, none; 1+, mild; 2+, moderate; 3+, moderate-severe; 4+, severe; scores of crescent formation and necrosis were doubled) were assigned for each of the above features and then added together to yield a final renal score. Changes were also assessed in terms of whether they were focal or diffuse. Vasculitis was judged as being either present or absent. 2.6. Statistical analysis All data were analyzed using Prism version 3.0 software (GraphPad, San Diego, CA). When a single treatment group was compared with its control, two-sample Student’s t or Wilcoxon rank-sum tests were used for parametric and nonparametric data, respectively. For multiple comparisons, one-way ANOVA followed by Tukey’s pairwise comparisons were used. Mann–Whitney nonparametric two-tailed U tests were utilized to test for significance between groups in single group comparisons, as in immunofluorescence scoring and pathology. Fisher’s exact probability tests (two-tailed) were utilized to test for significance between groups in single group comparisons, as in occurrence of vasculitis. Logrank analysis was used to compare trends in occurrence of albuminuria and animal survival. A value of p < 0.05 was considered statistically significant.
Fig. 1. Serum anti-dsDNA antibody levels in NZB/W F1 mice. CR2-fH, CR2-Crry and sCR2 treatment groups showed significantly reduced serum anti-dsDNA antibody levels compared to saline controls. Results are expressed as the mean ± SEM. n = 10 in the CR2-fH, CR2-Crry and sCR2 treatment groups, and n = 14 in the saline control group.
3. Results
C). Mice treated with either CR2-Crry or sCR2 showed a delayed onset of albuminuria (29 weeks), however, there was no statistically significant differences in over all levels of urinary albumin excretion or in the occurrence of moderate or severe albuminuria compared to the control group by the time of sacrifice. In contrast, mice treated with CR2-fH showed a significantly lower occurrence of moderate albuminuria (p = 0.031) and lower occurrence of severe albuminuria (p = 0.050) compared to the control group. Although there was a trend towards decrease albuminuria in the CR2-fH group compared to the CR2-Crry and sCR2 groups, the difference did not reach statistical significance. These results indicate that targeted selective alternative pathway inhibition therapy is an effective treatment for glomerular filtration barrier injury in NZB/W F1 mice.
3.1. Serum anti-dsDNA autoantibody levels
3.3. Renal deposition of immune complex (IgG) and C3
Anti-dsDNA antibodies are associated with lupus-like renal disease in NZB/W F1 mice and are an indicator of an autoimmune response (Stott et al., 1986). Serum anti-dsDNA antibody levels increased progressively and significantly in the control group after week 25. In contrast, mice treated with CR2-fH, CR2-Crry or sCR2 showed modest elevation of anti-dsDNA antibody levels in their sera (Fig. 1). The difference between anti-dsDNA antibody levels in the control group vs. the CR2-fH, CR2-Crry and sCR2 treated groups was significant at week 31 (p < 0.05 for CR2-fH and CR2-Crry, and p < 0.01 for sCR2).
Mice were sacrificed at 32 weeks and kidneys removed for pathological assessment. To assess glomerular IC (IgG) and C3 deposition, frozen kidney sections were stained with fluoresceinconjugated anti-mouse IgG or anti-mouse C3. There was no significant difference in glomerular IgG deposition between the control group and any treatment group, although there was a trend towards reduced glomerular IgG deposition levels in the CR2-fH, CR2-Crry, and sCR2 treated groups compared to the control group (Fig. 3A). This trend is consistent with the effect of these three proteins on anti-dsDNA antibody levels (Fig. 1). Glomerular C3 deposition, on the other hand, was significantly reduced in CR2fH, CR2-Crry, and sCR2 treated mice compared to controls (Fig. 3B) (p < 0.001, CR2-fH vs. saline; p = 0.016, CR2-Crry vs. saline; p = 0.002, sCR2 vs. saline).
3.2. Albuminuria Glomerulonephiritis is a major cause of death in NZB/W F1 mice, as it is in MRL/lpr mice and in human lupus. To determine the effect of complement inhibition on urinary protein excretion, and therefore renal function, we determined 24 h urinary albumin excretion levels starting at week 23. Control NZB/W F1 mice developed increasing albuminuria from 27 weeks of age (Fig. 2A), and 8 out of 14 mice (57%) developed moderate albuminuria (>1 mg/mouse/day) and half of the mice (7/14) developed severe albuminuria (>5 mg/mouse/day) by the time of sacrifice (Fig. 2B and
3.4. Renal pathology Kidney sections were stained with H&E and assessed by histological scoring for overall glomerular proliferative changes, crescent formation and necrosis, interstitial inflammation and incidence of vasculitis. NZB/W F1 mice in the control group exhibited diffuse glomerulonephritis, including cellular proliferation,
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Fig. 2. Albuminuria in NZB/W F1 mice. (A) Urinary albumin excretion levels in NZB/W F1 mice. Results are expressed as the mean 24 h albumin excretion (mg/mouse/day) ± SEM. (B and C) Albuminuria development over time (Kaplan–Meier curve) in NZB/W F1 mice. Number of mice excreting moderate ((B) >1.0 mg/mouse/day) or severe ((C) >5.0 mg/mouse/day) levels of albumin were recorded until the time of sacrifice (32 weeks). n = 10 for the CR2-fH, CR2-Crry and sCR treatment groups, and n = 14 for the saline control group.
Fig. 3. Assessment of glomerular IgG (A) and C3 (B) deposition by immunofluorescence microscopy. Sections were prepared from kidneys isolated from 32 week-old NZB/W F1 mice. Bars represent the mean. n = 9 in the CR2-fH treatment group, n = 8 in the CR2-Crry treatment group, n = 9 in the sCR2 treatment group, and n = 9 in the saline control group.
Table 1 Kidney pathology in NZB/W F1 mice. Group
Renal score (mean ± STD)a
Interstitial inflammation (mean ± STD)
Occurrence of vasculitis
CR2-fH (n = 9) CR2-Crry (n = 8) sCR2 (n = 9) Saline (n = 10)
6.0 ± 2.5 6.6 ± 3.0 5.7 ± 1.5 9.6 ± 5.6
1.3 ± 1.2 1.3 ± 0.6 1.1 ± 0.9 2.0 ± 1.4
1 out of 9 (11%) 1 out of 8 (13%) 0 out of 9 (0%) 2 out of 10 (20%)
p-Value CR2-fH vs. saline CR2-Crry vs. saline sCR2 vs. saline
0.036 0.140 0.075
0.252 0.276 0.185
1.000 1.000 0.474
Statistics
Mann–Whitney U test (two-tailed)
Mann–Whitney U test (two-tailed)
Fisher’s exact probability test (two-tailed)
The bold value is statistically significant. a The H&E kidney slides were graded for glomerular inflammation, proliferation, crescent formation, necrosis and vasculitis. Scores from 0 to 3+ (0, none; 1+, mild; 2+, moderate; 3+, severe; scores of crescent formation and necrosis were doubled) were assigned for each of these features and then added together to yield a final renal score.
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Fig. 4. Survival curves for NZB/W F1 mice treated with complement inhibitors. Mortality was recorded until the time of sacrifice (32 weeks). n = 10 for the CR2-fH, CR2-Crry and sCR treatment groups, and n = 14 in the saline control group.
inflammation, expansion, fibrocellular crescents and interstitial inflammation. CR2-fH treated mice had a significant decrease in pathologic features of disease compared to controls, with a reduction in mesangial expansion, glomerular inflammation, focal hypercellularity and crescent formation (reflected in the renal score, Table 1, p = 0.035). CR2-Crry and sCR2 treated mice showed a trend towards improved pathologic disease, but the difference did not reach statistical significance. Compared to control mice, all treated groups showed trends towards less interstitial inflammation and vasculitis; the differences, however, did not reach statistical significance (Fig. 4). 3.5. Survival Four out of 14 mice (28.6%) in the control group died before the time of sacrifice at 32 weeks, whereas only 1/10 mice (10.0%) died in each of the CR2-fH and sCR2 treated groups. This trend towards improvement in survival compared to control mice did not reach significance (p = 0.253 and 0.249, CR2-fH vs. saline and sCR2 vs. saline, respectively). As for the CR2-Crry treated group, there was no trend or statistically significant difference in survival benefit compared to the control group (p = 0.562). 4. Discussion CR2-Crry and CR2-fH are targeted to sites of C3 deposition by CR2-mediated binding to C3 proteolytic fragments (iC3b, C3dg, and C3d). CR2-Crry inhibits all complement pathways at the C3 level, whereas CR2-fH is specific for the alternative pathway (Huang et al., 2008). This paper addresses two therapeutic approaches in lupus; that of targeting complement inhibitors to sites of complement activation, and that of selective inhibition of the alternative pathway vs. inhibition of all pathways. The benefit of alternative pathway blockade in fB and fD deficient MRL/lpr mice vs. pan-inhibition of complement activation in C3 deficient mice was shown by an improvement in renal disease (i.e., albuminuria, renal score and mortality), as well as a decrease in autoantibody production as evidenced by reduction of serum anti-dsDNA antibody levels. To address efficacy of targeted inhibition of the alternative pathway in a clinical setting, we previously showed that treatment with CR2-fH significantly improved lupus-like renal disease in lupus-prone MRL/lpr mice (Sekine et al., 2011). Given that the pathogenic mechanisms underlying development of lupus vary by lupus prone strains, we considered it important to determine the efficacy of alternative pathway inhibition in a different lupus strain, and to compare alternative pathway inhibition vs. inhibition of all
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pathways. Note that the inhibitors used in this study act at the C3 activation step of the complement pathways, and potential effects of C1q or C4 opsonization on pathogenesis are not addressed. Compared to the saline control group, alternative pathway inhibition with CR2-fH resulted in significantly lower levels of serum anti-dsDNA antibody, glomerular C3 deposits, urinary albumin excretion, and pathologic scores of glomerulonephritis. Targeted inhibition of all complement pathways with CR2-Crry was also protective for some autoimmune manifestations, but was less effective overall than CR2-fH, in that it did not significantly reduce albuminuria or glomerulonephritis scores compared to the saline control. Consistent with the previous results of CR2-fH treated and fB and fD genotype knockout in MRL/lpr mice, the current data with NZB/W F1 mice indicate that inhibition of all complement pathways is less protective than selective inhibition of the alternative pathway for renal disease in two distinct murine models of SLE. These results further support the dual role of complement in lupus, and indicate that the alternative and classical pathways of complement play distinct roles in disease expression. As previously discussed (Sekine et al., 2011), the significant benefits of selectively inhibiting the alternative pathway may be related to the relative roles/contributions of the classical vs. alternative pathway in the handling of circulating immune complexes (ICs) and apoptotic cells. Both the alternative and classical pathways are involved in IC handling (Whaley, 1987), and therefore inhibiting both pathways has the potential to increase circulating IC levels and exacerbate disease. The classical pathway plays a key role in the clearance of apoptotic cells, and impaired clearance of the apoptotic material leads to increased inflammation and injury and progression of autoimmune manifestations. This conclusion is supported by the development of lupus in knockout mice in which clearance of apoptotic cells is impaired. There is the possibility that renal disease is being modulated as a result of a simple quantitative aspect to complement inhibition at the doses of each inhibitor used. However, both CR2-Crry and CR2-fH similarly reduce complement activation in kidneys in terms of C3 deposition, and we have previously shown that these CR2-targeted complement inhibitors have very little effect on systemic levels of complement activity at the doses used (Atkinson et al., 2005, 2008). Although the overall effect of CR2-fH on lupus like disease in NZB/W F1 mice was similar to that in MRL/lpr mice, there were some notable differences, possibly linked to different underlying pathogenic mechanisms in the two strains. CR2-fH in MRL/lpr mice resulted in a reduction of serum anti-dsDNA antibodies, while treatment with CR2-Crry had no effect on the levels of serum antidsDNA antibodies. In the current study using NZB/W F1 mice, total complement pathway inhibition with CR2-Crry resulted in a significant reduction of serum anti-dsDNA, and was similar to that seen with selective alternative pathway inhibition by CR2-fH or by sCR2 vehicle alone. This different outcome of CR2-Crry treatment for serum anti-dsDNA between MRL/lpr and NZB/W F1 mice may be due to a non complement mediated effect of the targeting molecule, sCR2, on autoimmune manifestations. Interferon (IFN)-␣, a type I IFN, is involved in the pathogenesis of SLE. IFN-␣ can detrimentally impact disease progression in NZB/W F1 mice, including autoantibody production, while it has a partial protective effect in MRL/lpr mice (Hron and Peng, 2004). IFN-␣ interacts with CR2 via its consensus repeat domains 1 and 2 (Kovacs et al., 2010). Thus, the potential sequestering and clearance of IFN-␣ by the CR2 moiety could impact disease expression, and may result in different outcomes for autoimmune manifestations, including the difference in anti-dsDNA antibody production between the strains (Hron and Peng, 2004). However, although NZB/W F1 mice treated with sCR2 had a significant reduction in serum anti-dsDNA antibody levels, and was similar to the reduction seen in mice treated
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with CR2-fH or CR2-Crry, we were unable to detect any differences in serum IFN␣ levels between treated and untreated mice (not shown). Additionally, or alternatively, serum CR2 may enhance maintenance of immune tolerance leading to decreased autoantibody production from B cells, as there is accumulating evidence showing an important role for CR2 in immune tolerance mechanisms that occur via interaction of C3b/C4b-decorated ICs with CR2 expressed on B cells (Holers and Boackle, 2004). The importance of this mechanism on disease may vary between strains. Indeed, it was previously shown that a significantly higher titer of antidsDNA antibodies and a more aggressive lupus-like renal disease was observed in Cr2−/− mice on both (C57BL/6 (B6) × 129)/lpr and B6/lpr backgrounds compared to Cr2+/+ controls (Prodeus et al., 1998; Wu et al., 2002). On the other hand, there was no significant differences in survival, anti-dsDNA antibody titers, or renal disease between Cr2−/− and Cr2+/+ lupus-prone MRL/lpr mice. Compared to Cr2+/+ MRL/lpr mice, Cr2−/− MRL/lpr mice did, however, show significantly reduced occurrence of renal vasculitis that maybe associated with significantly lower levels of IgG3 rheumatoid factor and total serum IgG3 (Boackle et al., 2004). Of note, there is evidence that defective Cr2 function in the NZB/W F1 background is linked to disease, and it is possible that sCR2 can rescue the phenotype in this strain. However, the fact that we previously found sCR2 to have a similar immune-modulatory effect in the MRL/lpr model of lupus (that is not linked to CR2), argues against the possibility that enhancing CR2 function improves disease. The current study provides evidence of CR2-mediated suppression of autoimmune manifestations, but further studies are required to define the precise mechanisms by which CR2 alone modulates autoimmunity in murine lupus. Paralleling the decrease in serum anti-dsDNA antibodies, all NZB/W F1 treated groups, including the group treated with sCR2, showed reduced glomerular IgG and C3 deposition compared to the saline control group. The difference in glomerular IgG deposition levels did not reach statistical significance in any group despite strong trends. Importantly, significant benefit for protection of the glomerular filtration barrier (i.e., reduction of albuminuria) and for protection from overall glomerular pathology (i.e., renal score) was only seen in mice treated with CR2-fH. Survival curves also showed reduced mortality in the CR2-fH treated group compared to the saline controls, but it did not reach statistical significance, probably due to early termination of survival analysis in favor of pathological analysis set at 32 weeks. Mortality was still less than 30% in the control group at the time of sacrifice.
5. Conclusion The current data indicate that selective inhibition of alternative pathway is more effective overall than total complement pathway inhibition in the NZB/W F1 murine model of lupus. The findings are in broad agreement with previous data generated in the MRL/lpr model of lupus. The current study also provides further evidence for the dual role of complement in lupus, and indicate distinct roles for the classical and alternative pathways of complement in the progression of the disease. Furthermore, the data indicate that the CR2 targeting moiety of the recombinant complement inhibitors modulates autoantibody production in murine lupus, and likely contributes to therapeutic efficacy. However, CR2-mediated suppression of autoimmune manifestations does not alone translate into significant improvements in the clinical features of lupus nephritis. Similar data in two distinct murine lupus models, each with differences in underlying pathogenic mechanisms, provide strong evidence that selective inhibition of the alternative pathway is a promising therapeutic approach to treat human lupus and
provides significant potential advantages over total complement inhibition.
Acknowledgements We thank Ting Ting Hsieh Kinser, Efrain Martinez and Emily Pauling for their expert technical assistance. This work was supported by a grant from the Department of Defense (W81 XWH07-1-0161).
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
The dual role of complement in the progression of renal disease in NZB/W F1 mice and alternative pathway inhibition Hideharu Sekine a , Phillip Ruiz b , Gary S. Gilkeson a , Stephen Tomlinson c,∗ a b c
Department of Medicine, Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston, SC 29425, USA Department of Pathology, University of Miami, School of Medicine, Miami, FL 33136, USA Department of Microbiology and Immunology, Darby Children’s Research Institute, Medical University of South Carolina, Charleston, SC 29425, USA
a r t i c l e
i n f o
Article history: Received 28 March 2011 Received in revised form 1 September 2011 Accepted 5 September 2011 Available online 14 October 2011 Keywords: Complement Lupus Factor H Kidney Autoimmunity
a b s t r a c t Complement plays a dual role in the progression of systemic lupus erythematosus since it has important protective functions, such as the clearance of immune complexes and apoptotic cells, but is also a mediator of renal inflammation. To investigate this balance in a clinically relevant setting, we investigated how targeted inhibition of all complement pathways vs. targeted inhibition of only the alternative pathway impacts immune and therapeutic outcomes in NZB/W F1 mice. Following onset of proteinuria, mice were injected twice weekly with CR2-fH (inhibits alternative pathway), CR2-Crry (inhibits all pathways at C3 activation step), sCR2 (C3d targeting vehicle) or saline. Sera were analyzed every 2 weeks for anti-dsDNA antibody levels, and urinary albumin excretion was determined. Kidneys were collected for histological evaluation at 32 weeks. Compared to the control group, all CR2-fH, CR2-Crry and sCR2 treated groups showed significantly reduced serum anti-dsDNA antibody levels and strong trends towards reduced glomerular IgG deposition levels. Glomerular C3 deposition levels were also significantly reduced in all three-treated groups. However, significant reductions of disease activity (albuminuria and glomerulonephritis) were only seen in the CR2-fH treated group. These data highlight the dual role played by complement in the pathogenesis of lupus, and demonstrate a benefit of selectively inhibiting the alternative complement pathway, presumably because of protective contributions from the classical and/or lectin pathways. The sCR2 targeting moiety appears to be contributing to therapeutic outcome via modulation of autoimmunity. Furthermore, these results are largely consistent with our previous data using the MRL/lpr lupus model, thus broadening the significance of these findings. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction The complement system plays an important role in host defense and immune regulation, but also plays a pathogenic role in many inflammatory and autoimmune diseases, including systemic lupus erythematosus (SLE or lupus) (Bao and Quigg, 2007; Chen et al., 2010). SLE is a prototypic autoimmune disease characterized by production of autoantibodies directed against a broad range of selfantigens, with formation and/or deposition of immune complexes (ICs) in targeted organs, followed by activation of the complement cascade leading to a local inflammatory process. The kidney is a major site of IC formation/deposition in lupus, and renal inflammation (i.e. lupus nephritis) is a major cause of morbidity and
Abbreviations: CR, complement receptor; SCR, short consensus repeat; SLE, systemic lupus erythematosus. ∗ Corresponding author. Tel.: +1 843 792 1450; fax: +1 843 792 2462. E-mail address: [email protected] (S. Tomlinson). 0161-5890/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2011.09.015
mortality, both in human SLE and in murine models of the disease. IC formation/deposition in glomeruli triggers activation of the classical pathway upon binding of C1q to an exposed antibody Fc region. C1r and C1s serine proteases, that are associated with C1q, are then activated and split C2 and C4 to form the enzyme complex C3 convertase, followed by C3 activation resulting in activation of the downstream anaphylotoxins (C3a and C5a), formation of the membrane attack complex (MAC, C5b–C9) and activation of the alternative complement pathway amplification loop through C3b. Paradoxically, patients with homozygous deficiencies of early components of the classical pathway (C1q, C1r/s or C4), have an increased prevalence of lupus and lupus-like disease (>80%) (Bao and Quigg, 2007; Walport, 2001). Homozygous deficiencies of C3, a protein that plays a pivotal role in all three complement activation pathways, is associated with membranoproliferative glomerulonephritis (Botto and Walport, 1993), but only rarely lupus (Pickering et al., 2000). This apparent contrasting role of complement in lupus is denoted “the lupus paradox”, and is explained by the fact that complement also plays a role in the clearance
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of apoptotic cells that are a potential source of self-antigens. In fact, C1q deficient mice have increased mortality and high titers of autoantibodies, with 25% of C1q−/− mice developing glomerulonephritis with glomerular IC deposits and multiple apoptotic bodies (Botto et al., 1998). C1q and C4 deficiency are also associated with the development/acceleration of lupus-like disease in mice on an autoimmune background (Einav et al., 2002; Mitchell et al., 2002), and C1q−/− and C4−/− mice (on 129/Sv × C57BL/6 background) exhibit an impaired ability to clear apoptotic cell bodies. Thus, these data support the hypothesis that the classical pathway plays a protective role in the development of lupus via its role in the clearance of apoptotic cells that otherwise provide a source of autoantigens to fuel autoimmune disease (Manderson et al., 2004). The alternative pathway is thought to play a key role in the inflammatory process in lupus nephritis. Indeed, previous studies have demonstrated a significant role for the alternative pathway in the development of renal disease in MRL/lpr mice, a murine strain that spontaneously develops lupus-like disease, including glomerulonephritis. For example, MRL/lpr mice deficient in either of the alternative pathway proteins factor B (fB) or factor D (fD) were protected from renal disease (Elliott et al., 2004; Watanabe et al., 2000). On the other hand, C3−/− MRL/lpr mice showed no improvement in pathologic renal disease and even worse albuminuria and increased glomerular IC deposits, likely due to decreased IC clearance by C3b (Sekine et al., 2001). Other complement inhibitors also showed protective effects for renal disease in MRL/lpr mice. The rodent membrane complement inhibitor Crry (complement receptor-related gene y) inhibits all complement pathways at the level of C3 by inhibiting C3 convertases, and administration of recombinant soluble forms of Crry (Crry-Ig (Quigg et al., 1998) and CR2-Crry (Atkinson et al., 2008)) provided protection against pathologic renal injury in MRL/lpr mice. However, treatment with the systemic inhibitor Crry-Ig resulted in increased levels of circulating ICs and no reduction in serum autoantibody levels compared to untreated controls. In contrast, treatment with CR2-Crry, which targets Crry to C3 breakdown products deposited at sites of complement activation, resulted in reduced serum autoantibody levels and no increase in circulating ICs. These different outcomes may be related to the systemic vs. localized nature of complement inhibition by CrryIg vs. CR2-Crry, respectively. Interestingly, the effect of CR2-Crry treatment on serum autoantibody levels, glomerular IC deposits and glomerulonephritis in MRL/lpr mice was observed only in mice receiving once a week administration (Atkinson et al., 2008), while mice receiving twice a week dosing did not demonstrate benefit in lupus like disease, possibly due to more complete and long lasting inhibition of complement. Based on these data, we recently investigated the effect of a new targeted inhibitor specific for the alternative pathway, CR2-fH, on renal disease in MRL/lpr mice. Compared to saline-injected controls, mice treated with CR2-fH had decreased serum autoantibodies, circulating IC, glomerular IC, C3 deposits, albuminuria, renal scores (pathological assessment of glomerulonephritis), vasculitis, and survival (Sekine et al., 2011). Collectively, the above studies indicate that targeted and selective inhibition of the alternative complement pathway is an effective treatment for lupus-like disease in MRL/lpr mice, and is more effective than inhibition of all complement pathways at the C3 level. However, there is a significant concern about different treatment outcomes in the different murine models of lupus given differences in pathogenesis between strains. Indeed, Fc␥RI and Fc␥RIII-deficient NZB/W F1 mice, another widely studied lupus-prone murine strain, had significantly decreased renal injury compared to littermate controls (Clynes et al., 1998), while MRL/lpr mice deficient in these same Fc receptors, still developed severe renal disease (Matsumoto et al., 2003). Interferon
(IFN)-␣, a type I IFN, is implicated in the pathogenesis of human SLE. However, it plays opposite roles in the development of lupus between murine lupus models. Treatment with IFN-␣ induces early lethal lupus nephritis in pre-autoimmune NZB/W F1 mice (Mathian et al., 2005), and type-1 IFN receptor deficiency reduces lupus-like disease (Santiago-Raber et al., 2003), indicating a pathogenic role for IFN-␣ in NZB/W F1 mice. In contrast, IFN-RI-deficient MRL/lpr mice developed worsened lupus-like disease, indicating a protective role of IFN-␣ in MRL/lpr (Hron and Peng, 2004). Thus, as in human lupus, efficacy of a therapy in one strain or patient, does not necessarily translate into effectiveness in all strains or patients. To determine whether selective alternative pathway inhibition is an effective treatment for lupus-like renal disease in a different lupus prone strain with different pathogenic mechanisms of disease (and thus also broaden potential clinical significance), we investigated and compared the effects of CR2-fH, CR2-Crry and sCR2 on lupus-like renal disease in NZB/W F1 female mice. 2. Materials and methods 2.1. Preparation and purification of CR2-fH, CR2-Crry and sCR2 The recombinant proteins CR2-fH, CR2-Crry and soluble CR2 (sCR2) were produced and purified as described previously (Atkinson et al., 2005; Huang et al., 2008). Proteins were expressed in Chinese hamster ovary (CHO) cells transfected with plasmids encoding either mouse CR2-fH, CR2-Crry or CR2, and purified from culture supernatants by anti-mouse CR2 (mAb 7G6) affinity chromatography as described (Atkinson et al., 2005; Huang et al., 2008). 2.2. Mice Female NZB/W F1 mice (#100008) were purchased from The Jackson Laboratory (Bar Harbor, ME). After the onset of renal disease at week 23 as determined by urinary protein excretion (>0.1 mg/mouse/day), the NZB/W F1 mice were randomized into four groups for biweekly treatment from weeks 23 to 31 as follows: (i) CR2-fH treatment group (n = 10, 0.4 mg twice a week), (ii) CR2-Crry treatment group (n = 10, 0.25 mg twice a week), (iii) sCR2 treatment group (n = 10, 0.18 mg twice a week) and (iv) control (saline) treatment group (n = 14, twice a week). CR2-fH, CR2-Crry, sCR2 and saline were administered by intraperitoneal (i.p.) injection. The dose and dosing frequency were selected to investigate the effect and potential relative benefits of targeted complement inhibition and were based on prior pharmacokinetic and efficacy experiments (Atkinson et al., 2005). All work with mice was approved by the Medical University of South Carolina Animal Protocols Review Board and was performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. 2.3. ELISA Measurements of serum anti-dsDNA antibody levels were determined by ELISA as previously described (Sekine et al., 2006). Pooled serum samples from 23-week-old MRL/lpr and C57BL/6 (B6) mice were used as positive and negative controls, respectively. 2.4. Urine albumin excretion Mice were placed in metabolic cages for 24 h urine collection every 2 weeks beginning at week 23. To prevent bacterial growth, antibiotics (ampicillin, gentamicin and chloramphenicol) were added to collection tubes. Urinary albumin excretion was determined by ELISA using a standard curve of known concentrations of mouse albumin (Bethyl Laboratories, Montgomery, TX) as
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previously described (Sekine et al., 2006). Results are expressed as milligrams of albumin per mouse per day. 2.5. Pathology At the time of sacrifice (32 weeks), the kidneys were removed. One kidney was snap frozen in liquid nitrogen and placed in OCT medium. 4-mm thick frozen sections were stained with fluorescein-conjugated anti-mouse IgG (MP Biomedical, Solon, OH) or anti-mouse C3 (MP Biomedical). Immunofluorescence slides were read blinded and graded on a scale of 0–3+ for fluorescence intensity (0, none; 1+, mild staining; 2+, moderate staining; 3+, high staining). The other kidney was fixed in 10% buffered formalin, embedded in paraffin, and sectioned before staining with hematoxylin and eosin (H&E). Slides were examined in a blinded fashion and graded for glomerular inflammation, proliferation, crescent formation and necrosis. Interstitial and tubular changes were also noted. Scores from 0 to 4+ (0, none; 1+, mild; 2+, moderate; 3+, moderate-severe; 4+, severe; scores of crescent formation and necrosis were doubled) were assigned for each of the above features and then added together to yield a final renal score. Changes were also assessed in terms of whether they were focal or diffuse. Vasculitis was judged as being either present or absent. 2.6. Statistical analysis All data were analyzed using Prism version 3.0 software (GraphPad, San Diego, CA). When a single treatment group was compared with its control, two-sample Student’s t or Wilcoxon rank-sum tests were used for parametric and nonparametric data, respectively. For multiple comparisons, one-way ANOVA followed by Tukey’s pairwise comparisons were used. Mann–Whitney nonparametric two-tailed U tests were utilized to test for significance between groups in single group comparisons, as in immunofluorescence scoring and pathology. Fisher’s exact probability tests (two-tailed) were utilized to test for significance between groups in single group comparisons, as in occurrence of vasculitis. Logrank analysis was used to compare trends in occurrence of albuminuria and animal survival. A value of p < 0.05 was considered statistically significant.
Fig. 1. Serum anti-dsDNA antibody levels in NZB/W F1 mice. CR2-fH, CR2-Crry and sCR2 treatment groups showed significantly reduced serum anti-dsDNA antibody levels compared to saline controls. Results are expressed as the mean ± SEM. n = 10 in the CR2-fH, CR2-Crry and sCR2 treatment groups, and n = 14 in the saline control group.
3. Results
C). Mice treated with either CR2-Crry or sCR2 showed a delayed onset of albuminuria (29 weeks), however, there was no statistically significant differences in over all levels of urinary albumin excretion or in the occurrence of moderate or severe albuminuria compared to the control group by the time of sacrifice. In contrast, mice treated with CR2-fH showed a significantly lower occurrence of moderate albuminuria (p = 0.031) and lower occurrence of severe albuminuria (p = 0.050) compared to the control group. Although there was a trend towards decrease albuminuria in the CR2-fH group compared to the CR2-Crry and sCR2 groups, the difference did not reach statistical significance. These results indicate that targeted selective alternative pathway inhibition therapy is an effective treatment for glomerular filtration barrier injury in NZB/W F1 mice.
3.1. Serum anti-dsDNA autoantibody levels
3.3. Renal deposition of immune complex (IgG) and C3
Anti-dsDNA antibodies are associated with lupus-like renal disease in NZB/W F1 mice and are an indicator of an autoimmune response (Stott et al., 1986). Serum anti-dsDNA antibody levels increased progressively and significantly in the control group after week 25. In contrast, mice treated with CR2-fH, CR2-Crry or sCR2 showed modest elevation of anti-dsDNA antibody levels in their sera (Fig. 1). The difference between anti-dsDNA antibody levels in the control group vs. the CR2-fH, CR2-Crry and sCR2 treated groups was significant at week 31 (p < 0.05 for CR2-fH and CR2-Crry, and p < 0.01 for sCR2).
Mice were sacrificed at 32 weeks and kidneys removed for pathological assessment. To assess glomerular IC (IgG) and C3 deposition, frozen kidney sections were stained with fluoresceinconjugated anti-mouse IgG or anti-mouse C3. There was no significant difference in glomerular IgG deposition between the control group and any treatment group, although there was a trend towards reduced glomerular IgG deposition levels in the CR2-fH, CR2-Crry, and sCR2 treated groups compared to the control group (Fig. 3A). This trend is consistent with the effect of these three proteins on anti-dsDNA antibody levels (Fig. 1). Glomerular C3 deposition, on the other hand, was significantly reduced in CR2fH, CR2-Crry, and sCR2 treated mice compared to controls (Fig. 3B) (p < 0.001, CR2-fH vs. saline; p = 0.016, CR2-Crry vs. saline; p = 0.002, sCR2 vs. saline).
3.2. Albuminuria Glomerulonephiritis is a major cause of death in NZB/W F1 mice, as it is in MRL/lpr mice and in human lupus. To determine the effect of complement inhibition on urinary protein excretion, and therefore renal function, we determined 24 h urinary albumin excretion levels starting at week 23. Control NZB/W F1 mice developed increasing albuminuria from 27 weeks of age (Fig. 2A), and 8 out of 14 mice (57%) developed moderate albuminuria (>1 mg/mouse/day) and half of the mice (7/14) developed severe albuminuria (>5 mg/mouse/day) by the time of sacrifice (Fig. 2B and
3.4. Renal pathology Kidney sections were stained with H&E and assessed by histological scoring for overall glomerular proliferative changes, crescent formation and necrosis, interstitial inflammation and incidence of vasculitis. NZB/W F1 mice in the control group exhibited diffuse glomerulonephritis, including cellular proliferation,
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Fig. 2. Albuminuria in NZB/W F1 mice. (A) Urinary albumin excretion levels in NZB/W F1 mice. Results are expressed as the mean 24 h albumin excretion (mg/mouse/day) ± SEM. (B and C) Albuminuria development over time (Kaplan–Meier curve) in NZB/W F1 mice. Number of mice excreting moderate ((B) >1.0 mg/mouse/day) or severe ((C) >5.0 mg/mouse/day) levels of albumin were recorded until the time of sacrifice (32 weeks). n = 10 for the CR2-fH, CR2-Crry and sCR treatment groups, and n = 14 for the saline control group.
Fig. 3. Assessment of glomerular IgG (A) and C3 (B) deposition by immunofluorescence microscopy. Sections were prepared from kidneys isolated from 32 week-old NZB/W F1 mice. Bars represent the mean. n = 9 in the CR2-fH treatment group, n = 8 in the CR2-Crry treatment group, n = 9 in the sCR2 treatment group, and n = 9 in the saline control group.
Table 1 Kidney pathology in NZB/W F1 mice. Group
Renal score (mean ± STD)a
Interstitial inflammation (mean ± STD)
Occurrence of vasculitis
CR2-fH (n = 9) CR2-Crry (n = 8) sCR2 (n = 9) Saline (n = 10)
6.0 ± 2.5 6.6 ± 3.0 5.7 ± 1.5 9.6 ± 5.6
1.3 ± 1.2 1.3 ± 0.6 1.1 ± 0.9 2.0 ± 1.4
1 out of 9 (11%) 1 out of 8 (13%) 0 out of 9 (0%) 2 out of 10 (20%)
p-Value CR2-fH vs. saline CR2-Crry vs. saline sCR2 vs. saline
0.036 0.140 0.075
0.252 0.276 0.185
1.000 1.000 0.474
Statistics
Mann–Whitney U test (two-tailed)
Mann–Whitney U test (two-tailed)
Fisher’s exact probability test (two-tailed)
The bold value is statistically significant. a The H&E kidney slides were graded for glomerular inflammation, proliferation, crescent formation, necrosis and vasculitis. Scores from 0 to 3+ (0, none; 1+, mild; 2+, moderate; 3+, severe; scores of crescent formation and necrosis were doubled) were assigned for each of these features and then added together to yield a final renal score.
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Fig. 4. Survival curves for NZB/W F1 mice treated with complement inhibitors. Mortality was recorded until the time of sacrifice (32 weeks). n = 10 for the CR2-fH, CR2-Crry and sCR treatment groups, and n = 14 in the saline control group.
inflammation, expansion, fibrocellular crescents and interstitial inflammation. CR2-fH treated mice had a significant decrease in pathologic features of disease compared to controls, with a reduction in mesangial expansion, glomerular inflammation, focal hypercellularity and crescent formation (reflected in the renal score, Table 1, p = 0.035). CR2-Crry and sCR2 treated mice showed a trend towards improved pathologic disease, but the difference did not reach statistical significance. Compared to control mice, all treated groups showed trends towards less interstitial inflammation and vasculitis; the differences, however, did not reach statistical significance (Fig. 4). 3.5. Survival Four out of 14 mice (28.6%) in the control group died before the time of sacrifice at 32 weeks, whereas only 1/10 mice (10.0%) died in each of the CR2-fH and sCR2 treated groups. This trend towards improvement in survival compared to control mice did not reach significance (p = 0.253 and 0.249, CR2-fH vs. saline and sCR2 vs. saline, respectively). As for the CR2-Crry treated group, there was no trend or statistically significant difference in survival benefit compared to the control group (p = 0.562). 4. Discussion CR2-Crry and CR2-fH are targeted to sites of C3 deposition by CR2-mediated binding to C3 proteolytic fragments (iC3b, C3dg, and C3d). CR2-Crry inhibits all complement pathways at the C3 level, whereas CR2-fH is specific for the alternative pathway (Huang et al., 2008). This paper addresses two therapeutic approaches in lupus; that of targeting complement inhibitors to sites of complement activation, and that of selective inhibition of the alternative pathway vs. inhibition of all pathways. The benefit of alternative pathway blockade in fB and fD deficient MRL/lpr mice vs. pan-inhibition of complement activation in C3 deficient mice was shown by an improvement in renal disease (i.e., albuminuria, renal score and mortality), as well as a decrease in autoantibody production as evidenced by reduction of serum anti-dsDNA antibody levels. To address efficacy of targeted inhibition of the alternative pathway in a clinical setting, we previously showed that treatment with CR2-fH significantly improved lupus-like renal disease in lupus-prone MRL/lpr mice (Sekine et al., 2011). Given that the pathogenic mechanisms underlying development of lupus vary by lupus prone strains, we considered it important to determine the efficacy of alternative pathway inhibition in a different lupus strain, and to compare alternative pathway inhibition vs. inhibition of all
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pathways. Note that the inhibitors used in this study act at the C3 activation step of the complement pathways, and potential effects of C1q or C4 opsonization on pathogenesis are not addressed. Compared to the saline control group, alternative pathway inhibition with CR2-fH resulted in significantly lower levels of serum anti-dsDNA antibody, glomerular C3 deposits, urinary albumin excretion, and pathologic scores of glomerulonephritis. Targeted inhibition of all complement pathways with CR2-Crry was also protective for some autoimmune manifestations, but was less effective overall than CR2-fH, in that it did not significantly reduce albuminuria or glomerulonephritis scores compared to the saline control. Consistent with the previous results of CR2-fH treated and fB and fD genotype knockout in MRL/lpr mice, the current data with NZB/W F1 mice indicate that inhibition of all complement pathways is less protective than selective inhibition of the alternative pathway for renal disease in two distinct murine models of SLE. These results further support the dual role of complement in lupus, and indicate that the alternative and classical pathways of complement play distinct roles in disease expression. As previously discussed (Sekine et al., 2011), the significant benefits of selectively inhibiting the alternative pathway may be related to the relative roles/contributions of the classical vs. alternative pathway in the handling of circulating immune complexes (ICs) and apoptotic cells. Both the alternative and classical pathways are involved in IC handling (Whaley, 1987), and therefore inhibiting both pathways has the potential to increase circulating IC levels and exacerbate disease. The classical pathway plays a key role in the clearance of apoptotic cells, and impaired clearance of the apoptotic material leads to increased inflammation and injury and progression of autoimmune manifestations. This conclusion is supported by the development of lupus in knockout mice in which clearance of apoptotic cells is impaired. There is the possibility that renal disease is being modulated as a result of a simple quantitative aspect to complement inhibition at the doses of each inhibitor used. However, both CR2-Crry and CR2-fH similarly reduce complement activation in kidneys in terms of C3 deposition, and we have previously shown that these CR2-targeted complement inhibitors have very little effect on systemic levels of complement activity at the doses used (Atkinson et al., 2005, 2008). Although the overall effect of CR2-fH on lupus like disease in NZB/W F1 mice was similar to that in MRL/lpr mice, there were some notable differences, possibly linked to different underlying pathogenic mechanisms in the two strains. CR2-fH in MRL/lpr mice resulted in a reduction of serum anti-dsDNA antibodies, while treatment with CR2-Crry had no effect on the levels of serum antidsDNA antibodies. In the current study using NZB/W F1 mice, total complement pathway inhibition with CR2-Crry resulted in a significant reduction of serum anti-dsDNA, and was similar to that seen with selective alternative pathway inhibition by CR2-fH or by sCR2 vehicle alone. This different outcome of CR2-Crry treatment for serum anti-dsDNA between MRL/lpr and NZB/W F1 mice may be due to a non complement mediated effect of the targeting molecule, sCR2, on autoimmune manifestations. Interferon (IFN)-␣, a type I IFN, is involved in the pathogenesis of SLE. IFN-␣ can detrimentally impact disease progression in NZB/W F1 mice, including autoantibody production, while it has a partial protective effect in MRL/lpr mice (Hron and Peng, 2004). IFN-␣ interacts with CR2 via its consensus repeat domains 1 and 2 (Kovacs et al., 2010). Thus, the potential sequestering and clearance of IFN-␣ by the CR2 moiety could impact disease expression, and may result in different outcomes for autoimmune manifestations, including the difference in anti-dsDNA antibody production between the strains (Hron and Peng, 2004). However, although NZB/W F1 mice treated with sCR2 had a significant reduction in serum anti-dsDNA antibody levels, and was similar to the reduction seen in mice treated
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with CR2-fH or CR2-Crry, we were unable to detect any differences in serum IFN␣ levels between treated and untreated mice (not shown). Additionally, or alternatively, serum CR2 may enhance maintenance of immune tolerance leading to decreased autoantibody production from B cells, as there is accumulating evidence showing an important role for CR2 in immune tolerance mechanisms that occur via interaction of C3b/C4b-decorated ICs with CR2 expressed on B cells (Holers and Boackle, 2004). The importance of this mechanism on disease may vary between strains. Indeed, it was previously shown that a significantly higher titer of antidsDNA antibodies and a more aggressive lupus-like renal disease was observed in Cr2−/− mice on both (C57BL/6 (B6) × 129)/lpr and B6/lpr backgrounds compared to Cr2+/+ controls (Prodeus et al., 1998; Wu et al., 2002). On the other hand, there was no significant differences in survival, anti-dsDNA antibody titers, or renal disease between Cr2−/− and Cr2+/+ lupus-prone MRL/lpr mice. Compared to Cr2+/+ MRL/lpr mice, Cr2−/− MRL/lpr mice did, however, show significantly reduced occurrence of renal vasculitis that maybe associated with significantly lower levels of IgG3 rheumatoid factor and total serum IgG3 (Boackle et al., 2004). Of note, there is evidence that defective Cr2 function in the NZB/W F1 background is linked to disease, and it is possible that sCR2 can rescue the phenotype in this strain. However, the fact that we previously found sCR2 to have a similar immune-modulatory effect in the MRL/lpr model of lupus (that is not linked to CR2), argues against the possibility that enhancing CR2 function improves disease. The current study provides evidence of CR2-mediated suppression of autoimmune manifestations, but further studies are required to define the precise mechanisms by which CR2 alone modulates autoimmunity in murine lupus. Paralleling the decrease in serum anti-dsDNA antibodies, all NZB/W F1 treated groups, including the group treated with sCR2, showed reduced glomerular IgG and C3 deposition compared to the saline control group. The difference in glomerular IgG deposition levels did not reach statistical significance in any group despite strong trends. Importantly, significant benefit for protection of the glomerular filtration barrier (i.e., reduction of albuminuria) and for protection from overall glomerular pathology (i.e., renal score) was only seen in mice treated with CR2-fH. Survival curves also showed reduced mortality in the CR2-fH treated group compared to the saline controls, but it did not reach statistical significance, probably due to early termination of survival analysis in favor of pathological analysis set at 32 weeks. Mortality was still less than 30% in the control group at the time of sacrifice.
5. Conclusion The current data indicate that selective inhibition of alternative pathway is more effective overall than total complement pathway inhibition in the NZB/W F1 murine model of lupus. The findings are in broad agreement with previous data generated in the MRL/lpr model of lupus. The current study also provides further evidence for the dual role of complement in lupus, and indicate distinct roles for the classical and alternative pathways of complement in the progression of the disease. Furthermore, the data indicate that the CR2 targeting moiety of the recombinant complement inhibitors modulates autoantibody production in murine lupus, and likely contributes to therapeutic efficacy. However, CR2-mediated suppression of autoimmune manifestations does not alone translate into significant improvements in the clinical features of lupus nephritis. Similar data in two distinct murine lupus models, each with differences in underlying pathogenic mechanisms, provide strong evidence that selective inhibition of the alternative pathway is a promising therapeutic approach to treat human lupus and
provides significant potential advantages over total complement inhibition.
Acknowledgements We thank Ting Ting Hsieh Kinser, Efrain Martinez and Emily Pauling for their expert technical assistance. This work was supported by a grant from the Department of Defense (W81 XWH07-1-0161).
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