The benefit of targeted and selective inhibition of the alternative complement pathway for modulating autoimmunity and renal disease in MRLlpr mice.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 63, No. 4, April 2011, pp 1076–1085 DOI 10.1002/art.30222 © 2011, American College of Rheumatology The Benefit of Targeted and Selective Inhibition of the Alternative Complement Pathway for Modulating Autoimmunity and Renal Disease in MRL/lpr Mice Hideharu Sekine,1 Ting Ting Hsieh Kinser,1 Fei Qiao,1 Efrain Martinez,1 Emily Paulling,1 Phillip Ruiz,2 Gary S. Gilkeson,1 and Stephen Tomlinson1 double-stranded DNA (anti-dsDNA) antibodies, and glomerular IgG and C1q deposition. Interestingly, sCR2 also significantly reduced the levels of anti-dsDNA antibodies and circulating ICs and reduced glomerular deposition of IgG, C1q, and C3, although there was no significant reduction in glomerulonephritis, proteinuria, or mortality. Conclusion. Targeted and selective inhibition of the alternative complement pathway is an effective treatment of murine lupus and is more effective than blockade of all pathways. The data demonstrate benefits to leaving the classical/lectin pathways intact and indicate distinct roles for the classical and alternative pathways of complement in disease progression. The sCR2-targeting vehicle contributes to therapeutic activity, possibly via modulation of autoimmunity. Objective. Complement has both protective and pathogenic functions in lupus due to a balance between its role in the clearance of immune complexes (ICs) and apoptotic cells and its role in inflammation. The classical pathway contributes to IC and apoptotic cell clearance, whereas the alternative pathway is a key mediator of renal inflammation. The aim of this study was to investigate the effect of a new targeted inhibitor of the alternative pathway, CR2-fH, on lupus-like renal disease in MRL/lpr mice. Methods. Mice were treated with either saline, CR2-fH, CR2-Crry (which inhibits all complement pathways), or soluble CR2 (sCR2; C3d-binding targeting vehicle). Sera were analyzed every 2 weeks for autoantibodies, circulating ICs, and C3. Urinary excretion of albumin was also determined, and kidneys were collected at 23 weeks for histologic evaluation. Results. Treatment with CR2-fH or CR2-Crry improved survival and significantly reduced proteinuria, glomerular C3 deposition, and the level of circulating ICs. CR2-fH, but not CR2-Crry, also significantly reduced glomerulonephritis, expression of serum anti– The complement system can be activated by 1 of 3 pathways (classical, lectin, and alternative) and provides important host defense, homeostasis, and immune regulatory functions. However, the complement system also plays a pathogenic role in many autoimmune and inflammatory diseases, including systemic lupus erythematosus (SLE) (1). SLE is the prototypic human autoimmune disease, and complement-mediated injury is initiated by immune complexes (ICs) formed by autoantibodies directed against a broad range of self antigens (2,3). The kidney is a major site of immune complex formation and/or deposition, and lupus nephritis is a major cause of morbidity and mortality, in both human SLE and murine models of lupus. There is, however, an apparent dual role for complement in the development and progression of lupus, as highlighted by the different roles of the classical and alternative complement pathways in the disease. Patients with homozygous deficiencies of early Supported by grants from the US Department of Defense (W81 XWH-07-1-0161) and the NIH (HL-082485). 1 Hideharu Sekine, MD, PhD, Ting Ting Hsieh Kinser, BSN, RN, Fei Qiao, MD, Efrain Martinez, RSII, BS, Emily Paulling, RSIII, VT, Gary S. Gilkeson, MD, Stephen Tomlinson, PhD: Medical University of South Carolina, Charleston; 2Phillip Ruiz, MD, PhD: University of Miami School of Medicine, Miami, Florida. Dr. Gilkeson owns stock or stock options in Taligen Therapeutics and has received consulting fees from that company (less than $10,000). Dr. Tomlinson owns stock or stock options in Taligen Therapeutics and has received consulting fees from that company (less than $10,000). Address correspondence to Stephen Tomlinson, PhD, Department of Microbiology and Immunology, Darby Children’s Research Institute, Medical University of South Carolina, Charleston, SC 29425. E-mail: firstname.lastname@example.org. Submitted for publication April 28, 2010; accepted in revised form December 23, 2010. 1076 ALTERNATIVE PATHWAY INHIBITION PROTECTS AGAINST LUPUS NEPHRITIS components of the classical pathway, such as C1 (C1q or C1r/s) or C4, have an increased incidence of lupus and lupus-like disease (⬎80%) (1,4). In contrast, homozygous deficiencies of C3, a protein that plays a pivotal role in all 3 complement activation pathways, is associated with membranoproliferative glomerulonephritis (5) but only rarely is associated with lupus (6). Studies in murine models also show a link between the classical pathway and autoimmune disease. Mice deficient in C1q have increased mortality and higher titers of autoantibodies, and glomerulonephritis with glomerular IC deposits and multiple apoptotic bodies develop in 25% of C1q-deficient mice (7). C1q deficiency and C4 deficiency are also associated with the development/acceleration of lupus-like disease in mice on an autoimmune background (8,9), and C1q⫺/⫺ and C4⫺/⫺ mice (on a 129/ Sv ⫻ C57BL/6 background) exhibit an impaired ability to clear apoptotic cell bodies. Taken together, these data are consistent with the hypothesis that the classical pathway provides 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 the disease process (10), although other hypotheses have been proposed (11). In contrast to the protective role of the classical pathway, there is strong evidence that the alternative pathway plays a key role in the development of lupus. In MRL/lpr mice, an autoimmune syndrome similar to human SLE develops spontaneously (12), and MRL/lpr mice deficient in either of the alternative pathway proteins, fB or fD, are protected from renal disease (13,14). In addition, various complement inhibitors are protective in murine models of lupus. Recombinant soluble forms of the mouse C3 inhibitor, Crry, provided protection against renal injury in MRL/lpr mice. These inhibitors, Crry-Ig (15), and CR2-Crry (16), also provided protection against skin/ear lesions and glomerular deposition of ICs and C3. However, only the targeted inhibitor, CR2-Crry, reduced glomerular inflammation, mortality, and autoantibody levels, and the levels of circulating ICs were significantly increased in MRL/lpr mice treated with Crry-Ig compared with those in mice treated with CR2-Crry. These effects of CR2-Crry were observed in mice treated once a week with the inhibitor. These different outcomes may be related to the systemic versus localized nature of complement inhibition by Crry-Ig and CR2-Crry, respectively. The CR2 moiety of the CR2-Crry fusion protein targets to iC3b, C3dg, and C3d, which are cell-bound activation fragments of C3 that are deposited at sites of complement 1077 activation. Complement inhibition with an anti-C5 monoclonal antibody is also protective in the NZB/NZW F1 model of lupus (17). Significantly, however, whereas C3 inhibition and alternative pathway deficiency are protective in MRL/lpr mice, C3 deficiency is not. In fact, compared with C3⫹/⫹ control mice, earlier and significantly greater albuminuria and increased glomerular IgG deposition was observed in C3⫺/⫺ MRL/lpr mice (18). Thus, total blockade of all complement pathways (as opposed to temporary and/or targeted blockade with inhibitors) was not protective and appeared to exacerbate disease. Collectively, the results of the above-mentioned studies suggest that selective inhibition of the alternative pathway will provide an effective therapeutic strategy for lupus, and that a targeted approach to complement inhibition has the potential to provide additional benefit with less immune suppression and toxicity. Here, we report on the characterization of CR2-fH, a recently described targeted complement inhibitor that is specific for the alternative pathway, in treatment of disease in the MRL/lpr mouse model of lupus. For clinical relevancy, treatment was begun after the onset of renal disease, and the effect of CR2-fH on immune modulation and disease progression was compared with the effect of CR2-Crry, an inhibitor of all complement pathways. MATERIALS AND METHODS Preparation and purification of CR2-fH, CR2-Crry, and soluble CR2 (sCR2). The recombinant proteins CR2-fH, CR2-Crry, and sCR2 were produced and purified as described previously (19,20). Mice. Female MRL/lpr mice (stock no. 000485) were purchased from The Jackson Laboratory. Of note, the mice used in this study were the strain recently recovered from the embryo archive that were cryopreserved in 1993 because of a progressive phenotype loss including reduced enlargement of brachial and mesenteric lymph nodes, decreased splenomegaly, and prolonged life spans (http://jaxmice.jax.org/strain/ 006825.html). MRL/lpr mice were randomized into 4 groups of 12–14 mice each for treatment from weeks 15 to 23 as follows: 1) CR2-fH treatment (0.4 mg twice weekly; n ⫽ 12), 2) CR2Crry treatment (0.25 mg twice weekly; n ⫽ 12), 3) sCR2 treatment (0.18 mg twice weekly; n ⫽ 12), and 4) control treatment group (saline twice weekly; n ⫽ 14). The inhibitor dosage was based on previously published dose-response data in a model of intestinal ischemia-reperfusion injury (19). CR2-fH, CR2-Crry, sCR2, and saline were administered by intraperitoneal injection. 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. 1078 SEKINE ET AL Enzyme-linked immunosorbent assay (ELISA). Measurements of the levels of serum anti–double-stranded DNA (anti-dsDNA) antibodies, circulating ICs, and C3 were determined by ELISA, as previously described (21). Pooled serum samples from 23-week-old MRL/lpr and C57BL/6 mice were used as positive and negative controls, respectively. Urinary albumin excretion. Mice were placed in metabolic cages for 24-hour urine collection every 2 weeks beginning at week 14. To prevent bacterial growth, antibiotics (ampicillin, gentamicin, and chloramphenicol) were added to the collection tubes. Urinary albumin excretion was determined by ELISA using a standard curve of known concentrations of mouse albumin (Bethyl Laboratories), as previously described (21). Results are expressed as milligrams of albumin per mouse per day. Assessment of pathology. At the time when the mice were killed (23 weeks), their kidneys were removed. One kidney was snap-frozen in liquid nitrogen and placed in OCT compound. Frozen sections (4 mm thick) were stained with fluorescein-conjugated anti-mouse IgG (MP Biomedicals), anti-mouse C1q (Cedarlane), or anti-mouse C3 (MP Biomedicals) and were visualized with a Nikon Eclipse 80i fluorescence microscope. Images of 10 glomeruli from each section were captured with a Nikon Digital Sight DS-5Mc digital camera at fixed exposure time. Average fluorescence intensity of the longest diameter per glomerulus was measured with Nikon NIS-Elements imaging software version 3.07. The average fluorescence intensity of 10 glomeruli in each section was then calculated. The other kidney was fixed in 10% buffered formalin, embedded in paraffin, and sectioned before staining with hematoxylin and eosin (H&E) or periodic acid–Schiff (PAS). Slides were examined in a blinded manner 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, and 4⫹ ⫽ severe; [scores for 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. Statistical analysis. All data were analyzed using Prism version 3.0 software (GraphPad). When a single treatment group was compared with its control, Student’s 2-sample t-test and Wilcoxon’s rank sum test were used for parametric and nonparametric data, respectively. For multiple comparisons, one-way analysis of variance followed by Tukey’s pairwise comparisons were used. Nonparametric Mann-Whitney 2-tailed U tests were used to test for significance between groups in single group comparisons (e.g., immunofluorescence scoring and pathology). Fisher’s 2-tailed exact tests were used to test for significance between groups in single group comparisons (e.g., occurrence of vasculitis). Log rank analysis was used to compare trends in occurrence of albuminuria and survival. P values less than 0.05 were considered significant. RESULTS Treatment of MRL/lpr mice. Groups of 12–14 female MRL/lpr mice were treated with either CR2-fH Figure 1. Serum anti–double-stranded DNA (anti-dsDNA) antibody (Ab) levels in MRL/lpr mice. Serum anti-dsDNA antibody levels were significantly reduced in mice treated with CR2-fH or soluble CR2 (sCR2) compared with controls after 20 weeks of age. Values are the mean ⫾ SEM results from 12 mice in each of the active-treatment groups and 14 mice in the control group. OD ⫽ optical density. (0.4 mg), CR2-Crry (0.25 mg), sCR2 (0.18 mg, molar equivalent of CR2-fH), or saline twice weekly via intraperitoneal injection from age 15 weeks to age 23 weeks, which was after the onset of renal disease as determined by urinary protein excretion (⬎0.1 mg/mouse/day). The dose and dosing frequency were selected to investigate the effect and potential relative benefits of targeted complement inhibition and were based on a pilot study using MRL/lpr mice and prior pharmacokinetic and efficacy experiments comparing CR2-Crry and CR2-fH in a model of ischemia-reperfusion injury (19). In the pilot study, administration of CR2-fH twice weekly to MRL/lpr mice at the time of proteinuria onset was more effective at reducing anti-dsDNA antibody levels and albuminuria than was weekly administration (although the sample number was low, and the differences did not reach significance [data not shown]). Serum levels of anti-dsDNA autoantibodies, circulating ICs, and C3. Production of anti-dsDNA antibodies, a T cell–dependent autoimmune response, is associated with lupus-like renal disease in MRL/lpr mice (22–24). There was a progressive rise in serum antidsDNA antibody levels in the control group after week ALTERNATIVE PATHWAY INHIBITION PROTECTS AGAINST LUPUS NEPHRITIS 14, whereas there was no significant increase in serum anti-dsDNA antibody levels in the groups receiving CR2-fH or sCR2 treatment (Figure 1). The difference between anti-dsDNA antibody levels in the control group compared with the groups treated with CR2-fH or sCR2 was significant from week 20 (P ⬍ 0.05, saline versus CR2-fH; P ⬍ 0.01, saline versus sCR2). AntidsDNA antibody levels in the group treated with CR2Crry were not significantly different from those in the control group. We assessed serum IC levels to determine whether decreased serum anti-dsDNA antibody levels correlated with circulating IC levels. There was a progressive rise in circulating IC levels in serum from the control group, similar to the increase seen in serum anti-dsDNA antibody levels (Figure 2A). From week 20, however, all treatment groups had significantly reduced levels of circulating ICs in their serum compared with the control group (P⬍ 0.05, saline versus CR2-fH and CR2-Crry; P ⬍ 0.01, saline versus sCR2). The levels of serum C3 in patients with SLE are known to show an inverse correlation with disease activity (as measured, for example, by anti-dsDNA antibody levels) due to its consumption following activation of complement. Serum C3 levels in the control group decreased as the mice aged (Figure 2B) and showed an inverse correlation with serum anti-dsDNA antibody levels (Figure 1). In contrast, serum C3 levels in CR2fH–treated mice were maintained, and were significantly higher than those in the control group by week 22 (P ⬍ 0.05). Serum C3 levels in the groups treated with CR2Crry or sCR2 were also higher than those in the control group, but the difference did not reach significance. Reduced C3 levels are considered indicative of complement activation, and as such, the significantly reduced complement activation seen in the group treated with CR2-fH, but not in the group treated with CR2-Crry (compared with controls) correlates with the lower levels of anti-dsDNA antibodies seen in CR2-fH–treated mice but not in CR2-Crry–treated mice (compared with controls). Albuminuria. Lupus-like glomerulonephritis in MRL/lpr mice is considered to be a major cause of death, as is glomerulonephritis in human lupus. Urinary protein (albumin) excretion in lupus is a feature of damage to the charge/size barrier between capillary lumen and urinary space in glomeruli (25). To determine the effect of complement inhibition on renal function, we measured 24-hour urinary albumin excretion starting at week 14. In control MRL/lpr mice, albuminuria increased beginning at 16 weeks of age, 1079 Figure 2. Serum circulating immune complex (IC) (A) and C3 (B) levels in MRL/lpr mice. A, Circulating IC levels were significantly reduced in mice treated with CR2-fH, CR2-Crry, or sCR2 compared with controls after 20 weeks of age. B, Serum C3 levels in CR2-fH– treated mice were maintained and were significantly higher than those in control mice after 20 weeks of age. Values are the mean ⫾ SEM results from 12 mice in each of the active-treatment groups and 14 mice in the control group. See Figure 1 for other definitions. and ⬎70% of mice (10 of 14) had severe albuminuria (⬎5 mg/mouse/day) by the time at which they were killed (Figure 3). In contrast, mice treated with either 1080 SEKINE ET AL Figure 3. Albuminuria in MRL/lpr mice. A, Urinary albumin excretion levels were significantly reduced in mice treated with CR2-fH or CR2-Crry compared with controls after 20 weeks of age. Values are the mean ⫾ SEM. B and C, Kaplan-Meier curves showing the development of moderate (albumin excretion ⬎1.0 mg/mouse/day) and severe (⬎5.0 mg/mouse/day) albuminuria. Values were recorded until the time at which the mice were killed (23 weeks). All results are from 12 mice in each of the active-treatment groups and 14 mice in the control group. sCR2 ⫽ soluble CR2. CR2-fH or CR2-Crry had significantly less albuminuria compared with controls, in terms of both lower urinary albumin excretion levels and a lower occurrence of moderate (excretion of ⬎1 mg/mouse/day) or severe (⬎5 mg/mouse/day) albuminuria (Figure 3). Notably, no mice treated with CR2-fH experienced the development of severe albuminuria. In mice treated with sCR2, albuminuria increased beginning at week 18, but showed only a trend toward reduced albumin excretion levels compared with the control group (P ⫽ 0.061). However, the number of sCR2-treated mice in which severe albuminuria developed was significantly decreased compared with controls (P ⬍ 0.05). Renal deposition of IgG and complement. To determine the mechanistic effect of treatment on renal disease, mice were killed at 23 weeks of age, and their kidneys were removed for assessment of pathology. To assess glomerular IC (IgG), C1q, and C3 deposition, frozen kidney sections were stained with fluorescein-conjugated anti-mouse IgG, anti-mouse C1q, or anti-mouse C3. There was no significant difference in glomerular IgG or C1q deposition levels between CR2-Crry–treated mice and controls (Figures 4A, B, and D). The glomerular IgG and C1q deposition levels in CR2-fH–treated mice were significantly reduced compared with those in controls (for glomerular IgG, P ⫽ 0.017, CR2-fH versus saline; for glomerular C1q, P ⫽ 0.003, CR2-fH versus saline). Mice treated with sCR2 also showed a trend toward reduced glomerular IgG deposition levels and significantly reduced glomerular C1q deposition levels compared with controls (for glomerular IgG, P ⫽ 0.051, sCR2 versus saline; for glomerular C1q, P ⫽ 0.010, sCR2 versus saline). These results are consistent with the effect of these 2 proteins on anti-dsDNA antibody levels, and anti-dsDNA antibodies represent the predominant specificity of immunoglobulins depositing in glomeruli in MRL/lpr mice (26) and patients with SLE (27). In contrast, glomerular C3 deposition was significantly reduced in the groups treated with CR2-fH, CR2-Crry, or sCR2 compared with controls (Figures 4C and 4D) (P ⫽ 0.001, CR2-fH versus saline, sCR2 versus saline, and CR2-Crry versus saline). Renal pathology. H&E-stained and PAS-stained kidney sections were assessed by histologic scoring for overall glomerular damage, crescent formation and necrosis, interstitial inflammation, and the incidence of vasculitis. As expected, MRL/lpr mice in the control group exhibited classic features of lupus renal disease, with diffuse glomerulonephritis including cellular proliferation, inflammation, glomerular expansion, fibrocellular crescents, interstitial inflammation, and vasculitis. CR2fH–treated mice, however, exhibited a significant im- ALTERNATIVE PATHWAY INHIBITION PROTECTS AGAINST LUPUS NEPHRITIS 1081 Figure 4. A–C, Assessment of glomerular IgG (A), C1q (B), and C3 (C) deposition by immunofluorescence (IF) microscopy; sections were prepared from the kidneys of 23-week-old MRL/lpr mice. Each symbol represents an individual mouse. Bars show the mean. D, Representative images. sCR2 ⫽ soluble CR2. Original magnification ⫻ 400. provement in glomerular features of disease, with a reduction in mesangial expansion, glomerular inflammation, focal hypercellularity, and crescent formation, as reflected by the renal score (P ⫽ 0.019 versus saline) (Table 1). There was no significant difference in interstitial inflammation between the control group and any Table 1. treatment group. Medium vessel vasculitis in the kidney is another pathologic feature of renal disease in MRL/lpr mice (28), and the frequency and intensity of arteritis were also significantly reduced in CR2-fH–treated mice (P ⫽ 0.044 versus saline) (Table 1). Although there was a trend toward improved glomerular features in mice Kidney pathology in MRL/lpr mice* Treatment group Renal score Interstitial inflammation Vasculitis incidence CR-2fH (n ⫽ 10) CR2-Crry (n ⫽ 10) sCR2 (n ⫽ 8) Saline (n ⫽ 7) 8.7 ⫾ 2.7† 10.1 ⫾ 2.9 9.4 ⫾ 2.5 12.3 ⫾ 2.3 2.6 ⫾ 1.0 2.8 ⫾ 0.4 3.0 ⫾ 0.0 2.7 ⫾ 0.5 5 (50)‡ 8 (80) 6 (75) 7 (100) * Values for the renal score and interstitial inflammation are the mean ⫾ SD. Values for vasculitis incidence are the number (%) of mice. Hematoxylin and eosin⫺stained kidney sections were graded for glomerular inflammation, proliferation, crescent formation, necrosis, and vasculitis. Scores from 0 to 4⫹ (0 ⫽ none, 1⫹ ⫽ mild, 2⫹ ⫽ moderate, 3⫹ ⫽ moderate-severe, and 4⫹ ⫽ severe [scores for crescent formation and necrosis were doubled]) were assigned for each of the above features and then added together to yield a final renal score. sCR2 ⫽ soluble CR2. † P ⫽ 0.019 versus saline, by Mann-Whitney 2-tailed U test. ‡ P ⫽ 0.044 versus saline, by Fisher’s 2-tailed exact test. 1082 SEKINE ET AL Figure 5. Survival curves for MRL/lpr mice treated with complement inhibitors. Mortality was recorded until the time at which the mice were killed (23 weeks). Results are from 12 mice in each of the active-treatment groups and 14 mice in the control group. sCR2 ⫽ soluble CR2. treated with CR2-Crry or sCR2 compared with control mice, the difference was not statistically significant. Survival. CR2-fH treatment significantly prolonged the survival of MRL/lpr mice. By 21 weeks of age, 50% mortality was observed among control mice, compared with 100% survival in mice treated with CR2-fH (P ⫽ 0.028). There was a strong trend toward improvement in CR2-Crry–treated mice, but the difference in survival compared with control mice did not reach significance (P ⫽ 0.081). There was no survival benefit for mice treated with sCR2 (Figure 5). Antibody response to the complement inhibitor fusion proteins. Although the fusion proteins are composed of mouse proteins, it is possible that the linking sequence or conformation of the fusion protein could elicit a neutralizing antibody response. However, we detected no evidence of an antibody response to either CR2-Crry or CR2-fH, as measured by ELISA using serum prepared from mice 4 weeks after the beginning of treatment (data not shown). DISCUSSION The complement inhibitors CR2-Crry and CR2-fH both function at the C3 activation step of the complement cascade, and both are targeted to sites of C3 deposition via their CR2 domain. Mouse Crry has cofactor activity for factor I–mediated cleavage of both C3b and C4b and has decay-accelerating activity for both classical and alternative pathway C3 convertases (29). The C5 convertase activities of Crry have not been investigated. The N-terminal domain of fH used in the CR2-fH construct has both decay-accelerating activity (displaces the Bb subunit) and cofactor activity for alternative pathway C3 and C5 convertases but does not control the activity of classical pathway C3 or C5 convertase (30). We previously demonstrated that CR2Crry inhibits all complement pathways, whereas CR2-fH is specific for the alternative pathway (19). In addition, we previously showed that CR2-Crry and CR2-fH have similar circulatory half-lives (8.8 and 8.7 hours, respectively) (19). We report here on the characterization of these 2 targeted inhibitors and also of the targeting domain alone (sCR2) in the MRL/lpr mouse model of lupus nephritis. Targeted inhibition of the alternative pathway with CR2-fH after the onset of proteinuria resulted in significant improvement in clinical features and a significant reduction in the number and severity of autoimmune manifestations. Targeted inhibition of all complement pathways with CR2-Crry was also protective but was less effective than CR2-fH in that it did not significantly reduce pathogenic glomerulonephritis, did not significantly improve survival, and had no significant effect on anti-dsDNA antibody and glomerular IgG or C1q deposition levels. The fact that total complement inhibition was less protective than selective inhibition of the alternative pathway provides further support for the dual role of complement in lupus and indicates that the alternative and classical pathways of complement play distinct roles in disease expression after onset. The significant benefits of selectively inhibiting the alternative pathway may be related, at least in part, to the relative roles/contributions of the classical pathway versus the alternative pathway in the handling of circulating ICs and apoptotic cells. The classical pathway plays an important role in the clearance of apoptotic cells, and impaired clearance of the apoptotic material that accumulates in murine lupus may lead to increased inflammation and injury and the progression of autoimmune manifestations. Whereas both the alternative and classical pathways are involved in IC handling (31), inhibiting both pathways has the potential to increase circulating IC levels and exacerbate disease. Although both treatment with CR2-fH and treatment with CR2Crry resulted in significantly lower levels of circulating ICs compared with those in control mice at week 22, CR2-Crry treatment did not significantly reduce antidsDNA antibody levels, unlike CR2-fH. Of note, in contrast to the effect of targeted complement inhibition, ALTERNATIVE PATHWAY INHIBITION PROTECTS AGAINST LUPUS NEPHRITIS it was previously shown that systemic complement inhibition with Crry-Ig resulted in markedly increased levels of circulating ICs in MRL/lpr mice compared with controls (32). The difference between targeted and untargeted complement inhibition on circulating IC levels may be related to IC clearance; the targeted inhibitors have a short circulatory half-life (19,20), and because serum complement activity is rapidly restored, complement is available to interact with nascent ICs. In addition to affecting clearance, the effect of the targeted inhibitors on circulating IC levels may be attributable to reduced IC formation as a result of decreased autoantibody production. In this context, we observed that the CR2targeting moiety itself significantly reduced circulating IC levels and at the same time lowered anti-dsDNA antibody titers in treated mice compared with control mice. Treatment with sCR2 had an effect similar to that of CR2-fH on autoantibody and circulating IC levels. Correlating with anti-dsDNA antibody levels, there were significant reductions in glomerular IgG and C1q deposition in mice treated with CR2-fH or sCR2 but not in CR2-Crry–treated mice. An association between glomerular IgG deposition and serum autoantibody levels would be consistent with a previous report indicating that anti-dsDNA antibodies represent a predominant specificity of the immunoglobulins depositing in the glomeruli of MRL/lpr mice and humans (26,27). In addition, glomerular C3 deposition was significantly lower in all treated groups compared with the control group. Importantly, glomerular C3 deposition in treated mice was inversely correlated with the serum C3 level, a known serologic marker of disease activity in lupus, together with serum anti-dsDNA antibody levels. Although we would expect reduced C1q deposition to correlate with reduced C3 activation and deposition in this model, we cannot rule out the possibility that the CR2 binding to C3d interfered with C3 detection in the kidney, even though we used a polyclonal anti-C3 antibody for detection. Although C1q deposition data do not specifically address classical (or lectin) pathway activation/inhibition in the kidney, reduced C1q deposition did correlate with reduced serum anti-dsDNA antibody levels and glomerular IgG deposition in mice treated with CR2-fH or sCR2. It is thus likely that these molecules indirectly reduce classical pathway activation by reducing IgG and thus C1q deposition. The reduced autoantibody/IC/C3 levels seen in treated mice may be attributable to more than one mechanism, because both CR2 and complement play multiple roles in modulating immunity. The current data 1083 certainly indicate an important role for the CR2targeting moiety. There is accumulating evidence showing an important role for CR2 in immune tolerance mechanisms that occur via interaction of C3b/C4bdecorated ICs with CR2 expressed on B cells (33–35). Thus, serum CR2 may enhance maintenance of immune tolerance, leading to decreased autoantibody production from B cells. Alternatively, a recent study characterized the interaction of interferon-␣ (IFN␣) with CR2 via its consensus repeat domains 1 and 2 (36). There is a strong association between IFN␣ and the pathogenesis of lupus in humans and in some murine models of lupus (37–39). Thus, the sequestering and clearance of IFN␣ by CR2 could impact disease expression, including autoantibody production (39), although blocking IFN␣ in MRL/lpr mice is not protective (39). Further studies are required to define the precise mechanisms by which CR2 alone modulated autoimmunity in MRL/lpr mice. What is clear, however, is that CR2-mediated suppression of autoimmune manifestations did not translate to significant improvements in the clinical features of lupus nephritis, and that active complement inhibition is a key feature of CR2-targeted therapy in MRL/lpr mice. Both CR2-fH and CR2-Crry were effective at reducing albuminuria, suggesting that both selective blockade of the alternative pathway and blockade of all pathways are protective against glomerular damage in situ (capillary endothelium, basement membrane, podocyte epithelium) after complement activation. However, alternative pathway blockade with CR2-fH clearly resulted in a better overall outcome, in that it also reduced the renal score, the occurrence of renal vasculitis, and mortality. With regard to the potential role of the alternative pathway in human lupus, measures of activation of the alternative pathway are performed in specific clinical situations as an assessment of disease. Studies by Buyon et al (40,41) demonstrated that serum levels of the Bb subunit are elevated during active disease, and that Bb subunit measurements are useful clinically in assessing disease activity in pregnant patients with lupus. Factor B deposition can also be demonstrated in the glomeruli in lupus nephritis (42,43). In a recent genomewide study and directed single-nucleotide polymorphism (SNP) analyses, there was an association between specific SNPs in the fH gene and the development of lupus (44). Although there are clearly differences in the human versus the murine complement systems and in human versus mouse lupus, the vast majority of pathogenic factors implicated in human lupus were first discovered in murine models or confirmed in murine models (45). 1084 SEKINE ET AL A previous study compared the effect of Crry-Ig (a systemic inhibitor) with that of CR2-Crry in MRL/lpr mice. Although Crry-Ig had an overall protective effect, CR2-Crry was superior in terms of reducing both autoimmune manifestations and clinical features of disease (and CR2-Crry also required a much lower dose and frequency of administration) (16). Here, we demonstrate significant benefits of CR2-fH compared with CR2-Crry. Of note, however, there are some apparent differences between our previous and current findings with regard to CR2-Crry therapy. Although CR2-Crry treatment was similarly protective against renal disease in the 2 studies, we previously observed that treatment with CR2-Crry significantly lowered anti-dsDNA antibody levels and did not significantly decrease circulating IC levels, in contrast to the current data. One important difference between the 2 studies is that the mice used in the current study were recently recovered from an embryo archive cryopreserved in 1993 because of a progressive phenotype loss. Compared with mice used in the previous study, these mice showed more progressive phenotypes of enlargement of brachial and mesenteric lymph nodes, splenomegaly, and reduced life spans (50% mortality at 21 weeks versus 50% mortality at 24 weeks and thereafter). Another difference between the current and previous studies is the dosing frequency. We previously administered CR2-Crry once weekly, whereas in the current study all treatments were given twice weekly. This dosing schedule was based on a pilot study indicating that twice-weekly treatment of mice with CR2-fH provided optimal benefit. In summary, the results of the current study show that selective inhibition of the alternative pathway is a promising therapeutic approach to treating lupus and provides significant potential advantages over total complement inhibition. In addition to the benefits of a targeted approach to complement inhibition, the CR2targeting moiety itself modulated autoimmunity, circulating IC levels, and serum C3 levels in the MRL/lpr model and likely contributed to therapeutic efficacy. The data also provide 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. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Tomlinson had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Sekine, Kinser, Qiao, Martinez, Paulling, Ruiz, Gilkeson, Tomlinson. Acquisition of data. Sekine, Kinser, Qiao, Martinez, Paulling, Ruiz, Gilkeson, Tomlinson. Analysis and interpretation of data. Sekine, Kinser, Qiao, Martinez, Paulling, Ruiz, Gilkeson, Tomlinson. REFERENCES 1. Bao L, Quigg RJ. Complement in lupus nephritis: the good, the bad, and the unknown. Semin Nephrol 2007;27:69–80. 2. Sekine H, Watanabe H, Gilkeson GS. Enrichment of antiglomerular antigen antibody-producing cells in the kidneys of MRL/MpJ-Fas(lpr) mice. J Immunol 2004;172:3913–21. 3. Kotzin BL. Systemic lupus erythematosus. 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