Abnormal Immune Complex Processing and Spontaneous Glomerulonephritis in Complement Factor H-Deficient Mice with Human 1 on This information is current as Erythrocytes of September 23, 2021. Jessy J. Alexander, Bradley K. Hack, Alexander Jacob, Anthony Chang, Mark Haas, Robert W. Finberg and Richard J. Quigg J Immunol published online 11 August 2010 Downloaded from http://www.jimmunol.org/content/early/2010/08/11/jimmun ol.1000683 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2010/08/11/jimmunol.100068 Material 3.DC1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 11, 2010, doi:10.4049/jimmunol.1000683 The Journal of Immunology

Abnormal Immune Complex Processing and Spontaneous Glomerulonephritis in Complement Factor H-Deficient Mice with Human on Erythrocytes

Jessy J. Alexander,* Bradley K. Hack,* Alexander Jacob,* Anthony Chang,† Mark Haas,‡ Robert W. Finberg,x and Richard J. Quigg*

Complement receptor 1 (CR1) on human erythrocytes (Es) and complement factor H (CFH) on rodent platelets perform , which is a function that allows the processing of immune complexes (ICs) bearing C3 by the mononuclear phagocyte system. Similar immune adherence occurs in the glomerular podocyte by CR1 in humans and CFH in rodents. As a model for human 2/2 IC processing, we studied transgenic mice lacking CFH systemically but with human CR1 on Es. These CR1huTg/CFH mice

spontaneously developed proliferative glomerulonephritis, which was accelerated in a chronic serum sickness model by active Downloaded from 2/2 immunization with heterologous apoferritin. ICs containing Ag, IgG and C3 bound to Es in CR1huTg/CFH mice. In this setting, there was increased IC deposition in glomeruli, attributable to the presence of CR1 on Es, together with the absence of CFH on platelets and podocytes. In the absence of plasma CFH, the accumulated ICs activated complement, which led to spontaneous and chronic serum sickness-induced proliferative glomerulonephritis. These findings illustrate the complexities of complement- dependent IC processing by blood cells and in the glomerulus, and the importance of CFH as a plasma complement regu-

lator. The Journal of Immunology, 2010, 185: 000–000. http://www.jimmunol.org/

he C system contains over 30 plasma and cell-associated (8). The binding of C-opsonized particles to human Es, in a reac- proteins, many of which are alike as a consequence of gene tion termed immune adherence (9), is attributable to CR1. - T duplication events during evolution (1). Activation through opsonized ICs bound to E CR1 are trafficked to cells of the mono- classical, alternative or lectin C pathways leads to the cleavage of nuclear phagocyte system for removal from the circulation (10, 11). C3 and C5 and generation of , C3b, C5a, and C5b-9. Comple- CR1 expression on Es is reduced in IC diseases, such as systemic ment is the first line of defense against some microorganisms and is lupus erythematosus, which may account for the tendency to de-

an integral component of innate and adaptive immune responses to posit ICs in sites, such as the renal glomerulus (12, 13). by guest on September 23, 2021 many others. Complement proteins are also important to clear im- Complement factor H (CFH) is an important fluid phase C reg- mune complexes (ICs) and material derived from apoptotic cells; in ulator (14, 15). CFH abnormalities can underlie human atypical doing so, they can shape the immune response to diverse Ags, hemolytic uremic syndrome, dense deposit disease (DDD), and including those derived from self and allogeneic tissue (1–3). age-related , with an ever-growing list of re- The regulators of complement activation proteins are composed sponsible (3, 16). As a general rule, DDD is attributable of short-consensus repeat domains of ∼60 aa, and they are highly to type I mutations leading to reduced functional plasma CFH and related within and between even distant species (4–6). The func- the ensuing unrestricted systemic alternative pathway activation, tional activities of these family members are attributable to their whereas atypical hemolytic uremic syndrome is attributable to type binding to C4 and C3 products (3, 7). Human complement receptor 1 II mutations clustering in the terminal SCRs, leading to impaired (CR1) (CD35) is a type 1 transmembrane protein containing 30 short ability of CFH to bind anionic sites, such as on endothelia, and to consensus repeats in its most common allele, which is present on provide local protection against complement activation (3). erythrocytes (Es), neutrophils, monocytes, eosinophils, B cells, When CFH is absent in CFH2/2 mice, animals develop a spon- some T cells, follicular dendritic cells, and glomerular podocytes taneous inflammatory glomerular disease (i.e., glomerulonephritis [GN]), which leads to the late death of ∼25% of mice of mixed *Department of Medicine and †Department of Pathology, University of Chicago, 129, C57BL/6, and DBA/2 backgrounds (17, 18). The genetic Chicago, IL 60637; ‡Department of Pathology and Laboratory Medicine, Cedars- x background clearly is important, because the 129 1 Sinai Medical Center, Los Angeles, CA 90048; and Department of Medicine, Uni- versity of Massachusetts Medical School, Worcester, MA 01655. has susceptibility loci (19). Histologically, glomeruli exhibit early Received for publication February 26, 2010. Accepted for publication July 2, 2010. C deposition, followed later by progressive IC deposits and glo- This work was supported by a grant from Kidneeds (to J.J.A.) and National Institutes merular hypercellularity, but without significant functional impair- of Health Grant R01DK041873 (to R.J.Q.). ment of glomerular filtration or permselectivity to protein passage 2/2 Address correspondence and reprint requests to Dr. Richard J. Quigg at the current (17, 18). Consistent with alternative pathway mediation, CFH address: Section of Nephrology, Division of Biological Sciences, University of Chi- mice with a coexistent factor B deficiency are protected from cago, 5841 South Maryland Avenue, MC5100, Chicago, IL 60637. E-mail address: 2/2 [email protected] disease (17). The spontaneous disease in CFH mice requires Abbreviations used in this paper: BUN, blood urea nitrogen; CFH, complement factor C5 (but not C6) and C factor I, presumably to generate proinflam- H; CR1, complement receptor 1; CSS, chronic serum sickness; DDD, dense deposit matory C5a and to create iC3b as ligand for b2-integrins on disease; E, erythrocyte; GN, glomerulonephritis; IC, immune complex; IF, immuno- inflammatory cells, respectively (18, 20, 21). Whereas C57BL/6 fluorescence. CFH2/2 mice have no evident glomerular disease, and wild type Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 C57BL/6 are resistant to developing GN in chronic serum sickness

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000683 2 GN IN CFH-DEFICIENT MICE WITH HUMAN CR1 ON Es

(CSS) induced by repetitive immunization with heterologous apo- (Sigma-Aldrich, Milwaukee, WI) given daily i.p. for 5 wk (22). Controls 2 2 2/2 / included CFH and CR1huTg mice immunized with the same schedule ferritin, all C57BL/6 CFH mice develop GN in CSS (22). 2/2 Complicating the interpretation of studies in CFH2/2 mice are our of apoferritin and CR1huTg/CFH mice receiving saline vehicle alone. At the conclusion of the 5-wk protocol, animals were euthanized for col- findings that CFH on platelets is responsible for immune adherence lection of serum, urine, and kidneys. In other experiments designed to (23). Interestingly, a similar CFH for CR1 switch appears to occur in identify apoferritin on Es and platelets by FACS and in kidneys by immu- the mouse podocyte as well (24). Hence, in CSS, CFH2/2 mice have nofluorescence (IF) microscopy, unlabeled apoferritin was replaced at dif- greater deposition of ICs, yet in these circumstances, GN does not ferent times with apoferritin labeled with Alexa 594 (Molecular Probes, Eugene, OR) to an average of five sites per molecule (26). For these ensue unless CfH is absent in plasma, illustrating the importance of experiments, animals were euthanized with blood and tissue collection fluid-phase, and presumably local glomerular, C regulation in this 4 h after the final i.p. dose of apoferritin. 2/2 model system (21). The spontaneous phenotype of CR1huTg/CFH mice was also ana- To examine a system potentially more relevant to humans, in which lyzed in a separate cohort of 17 mice, sacrificed at periodic intervals for E CR1 is responsible for IC processing, we used transgenic mice collection of serum, urine, and kidneys. In all experiments, mice were housed in a specific -free facility. All studies with animals were expressing human CR1 under a GATA1 promoter that drives ex- approved by the University of Chicago Animal Care and Use Committee. pression in erythroid cells (CR1huTg) (25). We generated CR1huTg/ CFH2/2 mice to simulate and study IC processing by E-CR1. Sur- Flow cytometry prisingly, although E-CR1 in these mice bound ICs, these ICs were Platelets and Es were isolated and suspended in PBS with 10 mg/ml BSA trafficked to the kidney, leading to spontaneous development of severe (PBS/BSA) as described previously (23). Human CR1 was detected on Es GN in all mice, which was accelerated in CSS. using mAb 7G9 (IgG2a) labeled with Alexa 488 (Molecular Probes) fol-

lowing the manufacturer’s directions. Mouse IgG and C3 were detected Downloaded from with Alexa 488-labeled anti-mouse IgG and anti-mouse C3 Abs (Cappel, Materials and Methods MP Biomedicals, Solon, OH). Individual Alexa 488–labeled Abs (100 ml Animal experiments of a 2 mg/ml stock) were added to 100 ml Es or platelets and incubated for 2/2 30 min at room temperature. Unbound Alexa 488-labeled Abs were re- CFH mice [from Drs. Matthew Pickering and Marina Botto, Ham- moved by washing twice with PBS/BSA. Es and platelets were selectively mersmith Hospital, London, U.K. (17)] were crossed with CR1huTg mice gated by FACS based on forward- and side-scatter profiles; 20,000 events bearing human CR1 on Es (25). Both mice were on the C57BL/6 genetic were counted using a FACSCalibur instrument (BD Biosciences, San Jose, . 2/2 background ( N12). The resulting CR1huTg/CFH mice lacked CFH on CA) and analyzed with FloJo software (Tree Star, Ashland, OR). http://www.jimmunol.org/ platelets and in plasma by FACS and Western blotting, respectively (22), whereas they had CR1hu on Es by FACS (25), which was not true of 2/2 Measurements from serum and urine CFH mice without the CR1huTg (data not shown). 2/2 The CSS model of IC-mediated GN was induced in CR1huTg/CFH Blood urea nitrogen (BUN) concentrations were detected with a Beckman mice by actively immunizing mice with 4 mg horse spleen apoferritin Autoanalyzer (Beckman Coulter, Fullerton, CA). Urinary by guest on September 23, 2021

2/2 2/2 FIGURE 1. Glomerular disease features in CFH and CR1huTg mice actively immunized with apoferritin. A, BUN values in individual CFH , 2/2 CR1huTg, and CR1huTg/CFH mice after 5 wk of active immunization with apoferritin; p = 0.061 by ANOVA. B, Scores for glomerulonephritis in 2/2 2/2 individual CFH , CR1huTg, and CR1huTg/CFH mice after 5 wk of active immunization with apoferritin. p , 0.001 by ANOVA followed by Tukey’s 2/2 2/2 pairwise comparisons among all three groups. Representative glomerular histopathology in CR1huTg (C), CFH (D), and CR1huTg/CFH mice (E, F) after 5 wk of active immunization with apoferritin. Arrows indicate intraglomerular inflammatory cells; asterisk indicates fibrocellular crescent (periodic acid-Schiff staining, original magnification 3400). The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

2/2 2/2 FIGURE 2. Features of humoral immune responses in CFH and CR1huTg mice actively immunized with apoferritin. CFH , CR1huTg,andCR1huTg/ 2/2 CFH mice were actively immunized for 5 wk with apoferritin; as controls, CR1huTg mice received saline instead of apoferritin. A, Flow cytometry for platelet- associated (left panel) and E-associated (right panel) IgG. Data are representative of experiments performed three times. B, Quantification of serum antiapoferritin IgG Abs by ELISA. Data from individual mice are shown. pp , 0.007 versus three other groups by ANOVA followed by Tukey’s pairwise comparisons. C, Presence of C3 in circulating immune complexes. Serum IgG was precipitated with protein G, followed by SDS-PAGE under reducing conditions and immunoblotting with anti-mouse C3 (green) and IgG (red) Abs. Each lane is from an individual mouse. Molecular weight standards are indicated on the left. concentrations were measured with a mouse albumin ELISA kit (Bethyl To detect C3 in ICs, 5 ml serum from individual animals diluted in 95 ml Laboratories, Montgomery, TX) and normalized to creatinine concen- PBS was added to a 20-ml slurry of protein G (Roche Applied Science, trations in the same urine (measured with Stanbio Laboratory Creatinine Indianapolis, IN). Samples were left overnight at 4˚C on a rotator. Tubes Procedure No. 0400; Boerne, TX). were centrifuged at 2500 3 g for 1 min at 4˚C. Beads were washed three Serum C3 was detected by Western blotting. Two microliters serum from times with 500 ml ice-cold PBS. To minimize background the supernatant individual animals was subjected to SDS-PAGE under reducing conditions, was completely removed after each wash. After the last wash, the pellet followed by electrophoretic transfer to a polyvinylidene fluoride membrane. was vortexed and heated at 90–100˚C for 5 min in 25 mlof13 Laemmli C3 was detected with goat anti-mouse C3 IgG (Cappel) followed by IRDye reducing sample buffer. Proteins were separated by SDS-PAGE and elec- 800 CW-labeled donkey anti-goat IgG (Rockland Immunochemicals, Gil- trophoretically transferred to a polyvinylidene fluoride membrane. Mem- bertsville, PA). Bound fluorescence was detected using an Odyssey Imager branes were incubated sequentially with goat anti-mouse C3 (Cappel), (LICOR Biosciences, Lincoln, NE). Of note is that with this approach, the IRDye 800 CW-labeled donkey anti-goat IgG, and Alexa 680-labeled goat anti-mouse C3 Ab appeared to react with the a-chain of mouse C3, but not anti-mouse IgG (Rockland, Gilbertsville, PA). Blots were washed with the b-chain (Supplemental Fig. 1). PBS/0.1% Tween-20, and fluorescence detected using an Odyssey Imager Anti-apoferritin IgG Abs were measured by ELISA as previously de- (LICOR Biosciences, Lincoln, NE). scribed (22). Polystyrene plates were coated with 5 mg/ml apoferritin. After blocking with 1% BSA, plates were incubated with serial dilutions Measurements from renal tissue of serum samples or anti-horse ferritin Ab (Jackson ImmunoResearch Laboratories, West Grove, PA), followed by HRP-conjugated anti- To evaluate pathologic renal changes, kidney tissues were fixed in 10% mouse IgG (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and buffered formalin, embedded in paraffin, and 4-mm sections were stained o-phenylenediamine peroxidase substrate (Sigma-Aldrich). The data were with periodic acid-Schiff. The extent of GN was graded by an observer presented as arbitrary units relative to a standard curve generated with masked to the origin of the slides on a scale of 0 to 4 (in increments of 0.5) antihorse ferritin Abs. as described previously (22). 4 GN IN CFH-DEFICIENT MICE WITH HUMAN CR1 ON Es

development of GN in every mouse. In contrast, wild type CFH- sufficient mice are uniformly resistant to GN in this model (22). To analyze how IC processing by E-associated CR1 might affect de- velopment of glomerular disease in this model, CSS was induced in 2/2 a group of five CR1huTg/CFH mice. As controls, CR1huTg (n = 4) and CFH2/2 mice (n = 3) were also immunized with apoferritin at the same time, and as an additional control CR1huTg mice (n =2) received vehicle alone. As is known to be the case, there was a marked reduction in serum C3 levels in CFH2/2 mice (17, 24). This acquired hypocom- plementemia was not affected by the presence of E-associated CR1 2/2 in CR1huTg/CFH mice, nor was it altered in the CSS model. CR1huTg mice had detectable C3, which did not appear to be af- fected by the CSS model (Supplemental Fig. 1). All CFH2/2 mice with CSS induced by active immunization with apoferritin for 5 wk developed GN characterized by pro- liferation within the mesangial and capillary regions and infiltration with inflammatory cells (Fig. 1B,1D). Despite inflammatory GN,

there was not impairment in glomerular filtration as determined by Downloaded from BUN measurements at the end of disease (Fig. 1A). These findings are consistent with our previous results in CFH2/2 mice with this model (22). Like wild type C57BL/6 mice, CR1huTg mice with intact CFH did not develop GN in this model (Fig. 1B,1C). Sur- 2/2 prisingly, CR1huTg/CFH mice with CSS had functional renal

impairment (BUN . 40 mg/dl in four of the five animals; Fig. 1A) http://www.jimmunol.org/ associated with severe proliferative GN (Fig. 1E,1F). All five animals in this study had GN scores $ 3.0 (Fig. 1B), indicating both endocapillary and extracapillary (i.e., crescentic) GN (Fig. 1F).

2/2 IC processing in CR1huTg/CFH mice with CSS

To analyze relevant features of the immune response in CR1huTg/ CFH2/2 mice, platelet- and E-associated IgG and serum antiapo- ferritin IgG Abs were measured in animals from the above studies. by guest on September 23, 2021 In these and subsequent experiments described below, studies were performed 4 h after the final i.p. dose of apoferritin, which presumably was absorbed from the peritoneum over this time. As shown in Fig. 2A, apoferritin-immunized CR1huTg but not CFH2/2 mice had platelet-associated IgG, which is consistent with the importance of platelet-associated CFH to bind ICs. Interestingly, 2/2 CR1huTg/CFH mice immunized with apoferritin had some FIGURE 3. Localization of C3 and apoferritin–IgG immune complexes in platelet-associated IgG, perhaps owing to platelet-associated CR1hu 2/2 2/2 glomeruli of CFH and CR1huTg/CFH mice. Mice were actively im- (25). Only CR1huTg mice immunized with apoferritin had E- 2/2 munized with apoferritin for 5 wk, which for the final 2 wk was as Alexa 594- associated IgG, with slightly less present in CR1huTg/CFH mice labeled apoferritin (red). Mouse C3 and IgG were detected with Alexa 488- relative to CR1huTg mice with intact CFH, possibly reflecting the 2/2 (green) and Alexa 350- (blue) conjugated primary Abs. In CFH mice, IgG acquired hypocomplementemia in the former, which could reduce and apoferritin were primarily in mesangial areas (arrows) distinct from C3 IC binding to CR1. By this approach, there was no detectable IgG on deposited in the peripheral capillary wall. In contrast, IgG and apoferritin were platelets or Es of CR1 Tg mice receiving vehicle as control. widely distributed in glomeruli of CR1 Tg/CFH2/2 mice, including in periph- hu hu All mice immunized with apoferritin, but not CR1 Tg con- eral capillary wall regions. These either lacked C3, as depicted by the arrow- hu trols receiving vehicle alone, had antiapoferritin IgG Abs in sera heads, or included C3, as was apparent in the region denoted with a solid white 2/2 line (original magnification 3600). (Fig. 2B). CFH mice had significantly higher levels of anti- apoferritin IgG compared with CR1huTg mice. Interestingly, CR1huTg For IF microscopy, 4-mm cryostat sections were fixed in ether-ethanol for mice had comparable antiapoferritin IgG levels irrespective of 10 min, followed by 95% ethanol for 20 min, and washed with PBS. The CFH status. Thus, the absence of CFH on platelets and the hypo- sections were stained with different combinations of Alexa-conjugated Abs to 2/2 mouse C3, IgG, IgA (Cappel, MP Biomedicals), and IgM (Sigma-Aldrich). complementemia in CR1huTg/CFH mice did not translate in- Slides were viewed with an Olympus (Melville, NY) BX-60 IF microscope. to greater circulating antiapoferritin IgG Abs relative to CR1huTg The glomerular distribution and semiquantitative score of staining intensity mice. In these analyses, it was not possible to exclude the (from0to4)wascompiledinamaskedmannerasdescribedpreviously(22). capture of antiapoferritin IgG in ICs. That the higher apparent antiapoferritin IgG in CFH2/2 mice could reflect the lack of Results platelet-associated CFH (and E-associated CR1) to bind anti- Glomerular disease features in CR1 Tg/CFH2/2 mice with hu apoferritin IgG in ICs (containing C3b) was examined in further CSS 2 2 studies. IgG in CFH / mouse serum contained C3 (presumably CSS induced by continued administration of heterologous apo- as the C3b a’110 chain), whereas there was no C3 associated with 2/2 2/2 ferritin leads to IC accumulation in glomeruli in CFH mice, with IgG in CR1huTg/CFH mice (Fig. 2C). These data support the The Journal of Immunology 5

finding that E-associated CR1 was capable of binding C3b- In glomeruli of CR1huTg mice, there was only a slight accumu- containing ICs. lation of Alexa 594-apoferritin with the mesangial regions, together 2/2 2/2 To further analyze IC handling by CR1huTg/CFH mice, var- with C3 and IgG (Fig. 5A). As expected, CFH mice had exten- iations of the CSS protocol were used in separate cohorts of sive deposits of C3 in the capillary wall, whereas Alexa 594- 2/2 2/2 CR1huTg/CFH and CFH mice. In initial experiments, 8-wk- apoferritin and IgG were centrally present within mesangial regions old mice (n = 3 per group) were immunized with unlabeled apo- of glomeruli (Fig. 5C). That the glomerular C3 was relatively in- ferritin as before for 3 wk. For the final 2 wk of the protocol, mice dependent of the abbreviated CSS protocol was shown by experi- were immunized with Alexa 594-labeled apoferritin. Glomerular ments with CFH2/2 mice receiving saline instead of apoferritin deposition of the labeled Ag was then evaluated together with over 3 wk followed by Alexa 594-apoferritin as in the other groups 2/2 2/2 IgG and C3. By this approach, CFH mice were found to have (Fig. 5B). Finally, in CR1huTg/CFH mice, there was extensive predominantly mesangial IgG- and apoferritin-containing ICs deposition of Alexa 594-apoferritin and IgG within glomeruli, (Fig. 3, white arrows), with some extension to the peripheral extending to the peripheral capillary walls (Fig. 5D,5E, arrows). 2/2 capillary wall. In both locations, these ICs tended not to colocal- Thus, these data expand on the findings that CR1huTg/CFH ize with the linear capillary wall staining for C3. In contrast, mice have abnormal IC metabolism, leading to short-term and long- 2/2 CR1huTg/CFH mice had greater quantities of IgG- and apofer- term IC deposition in glomeruli. ritin-containing ICs, which extended to the peripheral capillary wall (Fig. 3, white arrowheads), which in places colocalized with 2/2 C3 (Fig. 3, solid white line). Spontaneous development of GN in CR1huTg/CFH 2 2

/ Downloaded from In another set of experiments, 8-wk-old CR1huTg/CFH , Given the abnormal IC processing and development of severe GN 2/2 2/2 CFH , and CR1huTg mice (n = 3 per group) were immunized in CR1huTg/CFH mice with CSS, we studied a group of un- 2/2 with unlabeled apoferritin for 3 wk and then given a final immuni- manipulated 17 CR1huTg/CFH mice. Over time, these animals zation with Alexa 594-labeled apoferritin. Four hours later, ani- developed progressive renal functional impairment, characterized mals were sacrificed for analyses of platelet-, E-, and glomerular- by albuminuria (Fig. 6A) and azotemia, as documented by ele- associated apoferritin and C3. As shown in Fig. 4A,Alexa vated BUN values (Fig. 6B). Of the mice analyzed for over 20 wk,

594-apoferritin was present on platelets from CR1huTg but not two of five died (25 and 28 wk), and the remaining three had http://www.jimmunol.org/ 2/2 2/2 CFH mice. In CR1huTg/CFH mice, there was evidence for significant renal failure at 30 wk, with BUN values of 44, 60, and a small population of platelets bearing relatively large quantities of 90 mg/dl. Hematocrits remained $39 except for one 30-wk-old 2/2 apoferritin. Es from both CR1huTg groups, but not CFH mice, had animal with advanced renal failure (BUN = 90, in which the detectable apoferritin (Fig. 4B). Consistent with these data with hematocrit was 26). 2/2 2/2 apoferritin, both CR1huTg groups, but not CFH mice, had de- Histopathologically, CR1huTg/CFH mice developed diffuse tectable C3 on the E surface (Fig. 4D). Surprisingly, C3 was not endocapillary proliferative GN which became progressively more evident on platelets from any of the groups, including those with severe over time from 8 to 30 wk. At the later ages, in addition to intact CFH (i.e., CR1huTg; Fig. 4C). These data support the conclu- endocapillary proliferation, there was extracapillary proliferative by guest on September 23, 2021 sion that human CR1 on CR1huTg mouse Es bound ICs containing GN (GN scores $3.0). Fig. 7A–F show representative glomerular apoferritin and mouse C3, even in the relatively low-circulating C3 histopathology in the different age groups, and Fig. 7G shows GN 2/2 environment present in CR1huTg/CFH mice. scores for all animals. Only glomeruli were involved, except in the

2/2 FIGURE 4. ICs on CFH and CR1huTg mouse 2/2 platelets and Es in CSS. CR1huTg, CFH and 2/2 CR1huTg/CFH mice (n = 3 each) were actively immunized for 3 wk with unlabeled apoferritin, followed by a final dose of Alexa 594-labeled apo- ferritin. Four hours later, pooled platelets (A, C) and Es (B, D) from each group were analyzed by FACS for Alexa 594-labeled apoferritin (A, B) and mouse C3 (C, D). 6 GN IN CFH-DEFICIENT MICE WITH HUMAN CR1 ON Es

2/2 2/2 2/2 FIGURE 5. ICs in CFH and CR1huTg mouse glomeruli in CSS. CR1huTg (A), CFH (C), and CR1huTg/CFH mice (D, E; n = 3 each) were actively immunized for 3 wk with unlabeled apoferritin, followed by a final dose of Alexa 594-labeled apoferritin. As control, CFH2/2 mice receiving saline instead of apoferritin over the 3 wk period also received Alexa 594-labeled apoferritin (B). Four hours later, kidneys from each group were analyzed by IF for Alexa 594- apoferritin (red), mouse IgG (blue), and mouse C3 (green). The merged images of representative glomerular IF staining are shown. Arrows indicate Alexa 594- apoferritin and IgG in glomerular peripheral capillary walls (original magnification 3600). Downloaded from 30-wk-old mouse with renal failure, which also had tubulointer- CR1 on Es, but lacking CFH on platelets, would be physiologic stitial nephritis and arteritis. and allow more direct comparisons to humans. To our surprise, 2/2 Immunoreactants in glomeruli were also examined by IF mi- CR1huTg/CFH mice developed proliferative GN, which was croscopy. As is typical for CFH2/2 mice (17), there was consid- fatal in some, and could be accelerated in the CSS model. This erable C3 present in a linear staining pattern in peripheral finding contrasts with the spontaneous development of a form of 2/2 2/2 capillary walls of all CR1huTg/CFH mice (Fig. 8A–F). How- GN (i.e., DDD) in a minority of older CFH mice in a strain- http://www.jimmunol.org/ ever, there was also accumulation of IgG; initially, this was in dependent fashion (17). Thus, the presence of human CR1 on Es mesangial regions and appeared to be in locations distinct from of CFH2/2 mice proved to be detrimental, with development of the C3 staining (Fig. 8C,8D, arrows). At later ages, IgG extended progressive GN in these animals. This development was severe to peripheral capillary walls, including in thickened areas of the enough to lead to the death of animals over 25 wk of age. Thus, glomerular capillary wall (Fig. 8E,8F, arrowheads), which is rather than being physiologic, the presence of human CR1 on Es often associated with immunoreactive C3. There was minor promoted pathologic events in CFH2/2 mice. mesangial accumulation of IgM only at the later ages, and little to Regardless of the presence or absence of CFH, CR1huTg mice no staining for IgA in glomeruli in all animals (Fig. 8G). Thus, immunized with apoferritin had IgG, C3, and Ag associated with Es.

2/2 by guest on September 23, 2021 CR1huTg/CFH mice had abnormal IC processing independent It seems likely these were present in ICs containing mouse C3b bound of active immunization with exogenous Ag, leading to the spon- to human CR1. Consistent with a role for platelet-associated CFH as taneous development of GN. the immune adherence receptor, mice with intact CFH also had IgG and apoferritin Ag on platelets in the CSS model. Despite the rela- Discussion tively large amounts of IgG and Ag on these platelets, and that C3 In humans, CR1 on Es is the immune adherence receptor, re- could be detected on Es of CR1huTg mice, C3 was not detectable on sponsible for binding C3-containing ICs and properly transporting platelets in these mice, for which we do not have a clear explanation. them to the mononuclear phagocyte system (8–11). In contrast, In the CSS model, circulating antiapoferritin IgG Abs were greater in rodents rely on platelets for this function, which can be attrib- mice lacking both systemic CFH and E CR1, whereas they were uted to CFH (23). Thus, we hypothesized that creating mice with equivalent in mice with E CR1 regardless of whether CFH was

2/2 FIGURE 6. Development of biochemical features of renal disease over time in CR1huTg/CFH mice. Shown is albuminuria (A) and BUN (B)in 2/2 CR1huTg/CFH mice over time. Each symbol represents data from an individual mouse. The Journal of Immunology 7

2/2 FIGURE 7. Progressive development of glomerulonephritis over time in CR1huTg/CFH mice. Shown is representative histologic analysis in in- 2/2 2/2 dividual CR1huTg/CFH mice at 4 (A), 8 (B), 15 (C), 19 (D), and 30 (E, F) wk of age, and GN scores (G) in all CR1huTg/CFH mice over time. Each symbol represents data from an individual mouse. The arrows indicate intraglomerular inflammatory cells, and asterisk indicates a fibrocellular crescent Downloaded from (periodic acid-Schiff staining, original magnification 3400). present. Moreover, free IgG–C3b complexes were present only in the chimeric CFH2/2 mice support the conclusion that excessive IC sera of CFH2/2 mice. These data further support a role for E CR1 in deposition in glomeruli alone does not lead to inflammation or GN. antiapoferritin IgG handling in the CSS model. In these studies, excessive glomerular IC deposition occurred be- 2/2 CR1huTg/CFH mice had excessive IgG-containing ICs in cause of absent platelet CFH; however, GN did not result because http://www.jimmunol.org/ glomeruli spontaneously and in the CSS model. In the former in- plasma CFH was present (21). Moreover, even limited IC de- stance, the Ag could not be characterized, whereas in the latter it position occurring in mice with platelet CFH resulted in GN in clearly contained apoferritin. This was not simply a consequence of animals in which plasma CFH was absent. CR1 on Es, because CR1huTg mice also had E-associated IgG, Ag, Our short-term studies following labeled apoferritin Ag in the CSS and C3—presumably together in ICs. Hoewever, unmanipulated model provided further evidence for abnormal IC handling by mice 2/2 2/2 mice did not have glomerular ICs (data not shown) and only mild lacking CFH (i.e., CFH and CR1huTg/CFH mice). In CR1huTg 2/2 mesangial deposits in CSS, which is comparable to wild type mice. mice, ICs were present on both platelets and Es. In CR1huTg/CFH Interestingly, CFH2/2 mice lacking immune adherence receptors mice, they were present primarily on Es, and in CFH2/2 mice, they by guest on September 23, 2021 on either platelets or Es also had excessive glomerular ICs relative were absent from both platelets and Es. CR1huTg mice had only mild to wild type controls (both spontaneously and in CSS), but this was deposition of ICs within the glomerular mesangium, whereas CFH2/ 2/2 2 of a magnitude less than apparent in CR1huTg/CFH mice. Thus, mice had quantitatively greater IC deposits. Overall, findings these altered IC handling relative to the glomerulus required that human are consistent with our previous data showing that platelet-associated 2/2 CR1 be present on Es and CFH be absent systemically. CFH is necessary for IC processing (21). Yet, in CR1huTg/CFH 2/2 2/2 As occurs in CFH mice (17), young CR1huTg/CFH mice mice, there was substantially greater glomerular IC deposition with had substantial C3 in the glomerular capillary wall. Early in the extension to the peripheral capillary wall. This deposition occurred 2/2 spontaneously occurring disease in CR1huTg/CFH mice, ICs despite IC binding to Es in these mice. Thus, only in the situation in tended to be present in mesangial regions that did not have C3. With which CR1 was present on Es and CFH was absent systemically, did time, ICs progressively accumulated in the peripheral capillary excessive glomerular deposition of ICs result. wall, which initially may have replaced C3 in these areas. Yet, in Likely because mouse C3 has regions conserved with human C3, 2/2 both spontaneous GN in older CR1huTg/CFH mice and in the human CR1 effectively binds mouse C3b in rosetting assays (27) CSS model, glomerular ICs also contained adjacent C3 staining. and can bind mouse iC3 in a fluid-phase system, albeit ∼5-fold less Overall, we would argue the development of GN in CR1huTg/ than it does human iC3 (28). Thus, in the CSS model, CR1huTg mouse CFH2/2 mice was attributable to increased ICs in glomeruli, which Es had surface C3, presumably as C3b in ICs. Because of unrestricted activated C in an unrestricted C fashion because CFH was absent alternative pathway C activation, CFH2/2 mice have low plasma C3 2/2 from plasma. Our previous studies with CSS in bone marrow levels (17, 24), which was also true of CR1huTg/CFH mice. Yet,

FIGURE 8. Progressive accumulation of IgG in 2/2 glomeruli over time in CR1huTg/CFH mice. Shown are representative IF micrographs in individual 2/2 CR1huTg/CFH mice at 4 (A), 8 (B), 15 (C), 19 (D), and 30 (E–G) wk of age. A–F, Double staining for C3 (green) and IgG (red) are shown, while in G, staining was for IgA (green) and IgM (red). The white arrows and arrowheads indicate IgG in mesangial regions and pe- ripheral capillary walls, respectively. Original magnifi- cation 3200 (A–E, G) and 3600 (F). 8 GN IN CFH-DEFICIENT MICE WITH HUMAN CR1 ON Es

2/2 C3 appeared to be activated on ICs in CR1huTg/CFH mice, pre- podocytes. The accumulated ICs were proinflammatory in the ab- sumably reflecting the balance of low C3 available for classical path- sence of circulating CFH, which led to spontaneous and CSS- way activation, yet enhanced amplification through the alternative C induced severe proliferative GN. pathway, as also appears to occur in glomeruli in mice lacking plasma CFH (21). Related to this is the finding of lower quantities of E- Acknowledgments 2/2 associated C3 in CR1huTg/CFH mice compared with CR1huTg We thank Dr. Marina Botto and Dr. Matthew Pickering (Hammersmith Hos- mice. These data support the conclusion that CR1 on mouse Es in pital, London, U.K.) for providing CFH2/2 mice, Dr. Ron Taylor for the 2/2 CR1huTg/CFH micewas capable of binding mouse C3b- gift of Abs and for many helpful discussions, and Rebecca Alexander for containing ICs from the circulation. technical assistance. Beyond their ability to bind C3b, CFH and CR1 are both decay accelerators and cofactors for factor I-mediated inactivation of Disclosures C3-bearing alternative pathway convertases. Human CR1 can be The authors have no financial conflicts of interest. an effective complement inhibitor in mice in vitro and in vivo (29). Whether this is due to decay-accelerating and/or cofactor effects is not clear. It has also been shown that mouse factor I is References 1. Walport, M. J. 2001. Complement. First of two parts. N. Engl. J. Med. 344: compatible with human CR1 on Es for the cleavage of human C4b 1058–1066. (30). Thus, the existing data provide strong evidence that human 2. Schulze, C., L. E. Munoz, S. Franz, K. Sarter, R. A. Chaurio, U. S. Gaipl, and CR1 can bind mouse C3b, which is further supported by our M. Herrmann. 2008. Clearance deficiency—a potential link between infections and autoimmunity. Autoimmun. Rev. 8: 5–8. Downloaded from present studies, whereas the explanation of whether human CR1 3. Zipfel, P. F., and C. Skerka. 2009. Complement regulators and inhibitory pro- on mouse Es bearing C3b-containing ICs can recruit mouse factor teins. Nat. Rev. Immunol. 9: 729–740. I to lead to cleavage of C3b is not clear. C3 being present on CR1- 4. Barlow, P. N., M. Baron, D. G. Norman, A. J. Day, A. C. Willis, R. B. Sim, and I. D. Campbell. 1991. Secondary structure of a complement control protein transgenic mouse Es, but not CFH-bearing platelets, suggests that module by two-dimensional 1H NMR. Biochemistry 30: 997–1004. the functions of CR1 and CFH are dissimilar in these two sites. 5. Barlow, P. N., D. G. Norman, A. Steinkasserer, T. J. Horne, J. Pearce, Beyond their species compatibilities, CFH does not effectively P. C. Driscoll, R. B. Sim, and I. D. Campbell. 1992. Solution structure of the fifth repeat of factor H: a second example of the complement control protein module. bind C4b (as in ICs) or serve as factor I cofactor for cleavage Biochemistry 31: 3626–3634. http://www.jimmunol.org/ of iC3b to C3dg. Thus, overall there are a number of reasons why 6. Aslam, M., J. M. Guthridge, B. K. Hack, R. J. Quigg, V. M. Holers, and human CR1 on mouse Es is not physiologically relevant to mouse S. J. Perkins. 2003. The extended multidomain solution structures of the com- plement protein Crry and its chimeric conjugate Crry-Ig by scattering, analytical CFH on platelets. In fact, our data would be consistent with this ultracentrifugation and constrained modelling: implications for function and conclusion, given the excessive glomerular deposition of ICs (and therapy. J. Mol. Biol. 329: 525–550. 7. Morgan, B. P., and C. L. Harris. 1999. Regulation in the activation pathways. In development of GN) spontaneously and in CSS in CR1huTg/ 2/2 Complement Regulatory Proteins. Academic Press, San Diego, CA. p. 41–136. CFH mice. These findings raise interesting aspects for further 8. Khera, R., and N. Das. 2009. Complement Receptor 1: disease associations and study in this model system. therapeutic implications. Mol. Immunol. 46: 761–772. Although Es in CR1 Tg/CFH2/2 mice bound ICs, these animals 9. Nelson, D. S. 1963. Immune adherence. Adv. Immunol. 3: 131–180. hu 10. Medof, M. E., K. Iida, C. Mold, and V. Nussenzweig. 1982. Unique role of the 2/2 by guest on September 23, 2021 had excessive glomerular ICs relative to CR1huTg and CFH mice. complement receptor CR1 in the degradation of C3b associated with immune The lack of plasma CFH did not appear to substantially affect IC complexes. J. Exp. Med. 156: 1739–1754. binding to Es in CR1 Tg/CFH2/2 mice. Moreover, the presence of 11. Hebert, L. A. 1991. The clearance of immune complexes from the circulation of hu man and other primates. Am. J. Kidney Dis. 17: 352–361. human CR1 on mouse Es alone could not account for the excessive 12. Iida, K., R. Mornaghi, and V. Nussenzweig. 1982. Complement receptor (CR1) glomerular ICs. Whereas the lack of CFH on platelets appears to deficiency in erythrocytes from patients with systemic lupus erythematosus. contribute to glomerular IC deposition (21), there was a marked dif- J. Exp. Med. 155: 1427–1438. 2/2 13. Walport, M. J., G. D. Ross, C. Mackworth-Young, J. V. Watson, N. Hogg, and ference in glomerular ICs in CR1huTg/CFH mice compared with P. J. Lachmann. 1985. Family studies of erythrocyte complement receptor type 1 CFH2/2 mice. Thus, the lack of plasma and platelet CFH and the levels: reduced levels in patients with SLE are acquired, not inherited. Clin. Exp. 2/2 Immunol. 59: 547–554. presence of human CR1 in CR1huTg/CFH mice were not complete 14. Whaley, K., and S. Ruddy. 1976. Modulation of the alternative complement explanations for our findings of excessive glomerular ICs. pathways by b 1 H . J. Exp. Med. 144: 1147–1163. Besides Es, human glomerular podocytes use CR1 as an immune 15. Weiler, J. M., M. R. Daha, K. F. Austen, and D. T. Fearon. 1976. Control of the amplification convertase of complement by the plasma protein beta1H. Proc. adherence receptor (31, 32) for reasons that have largely been ob- Natl. Acad. Sci. USA 73: 3268–3272. scure (33). As with Es, rodent podocytes appear to replace CR1 with 16. Saunders, R. E., C. Abarrategui-Garrido, V. Fre´meaux-Bacchi, E. Goicoechea de CFH (34). Our previous studies using a renal transplantation ap- Jorge, T. H. Goodship, M. Lo´pez Trascasa, M. Noris, I. M. Ponce Castro, 2/2 G. Remuzzi, S. Rodrı´guez de Co´rdoba, et al. 2007. The interactive Factor H- proach in CFH mice have shown that podocyte CFH facilitates atypical hemolytic uremic syndrome database and website: update and the transcapillary passage of ICs in glomeruli (24). Interestingly, the integration of membrane cofactor protein and Factor I mutations with structural neonatal FcR is also present on human and mouse podocytes, where models. Hum. Mutat. 28: 222–234. 17. Pickering, M. C., H. T. Cook, J. Warren, A. E. Bygrave, J. Moss, M. J. Walport, and it clears IgG from the glomerular capillary wall (35). An attractive M. Botto. 2002. Uncontrolled C3 activation causes membranoproliferative glomer- explanation for our findings is that glomerular-deposited ICs are not ulonephritis in mice deficient in complement factor H. Nat. Genet. 31: 424–428. cleared appropriately in the absence of podocyte CFH. 18. Pickering, M. C., J. Warren, K. L. Rose, F. Carlucci, Y. Wang, M. J. Walport, 2/2 H. T. Cook, and M. Botto. 2006. Prevention of C5 activation ameliorates In summary, our studies concentrated on CR1huTg/CFH mice, spontaneous and experimental glomerulonephritis in factor H-deficient mice. which had human CR1 on Es, yet lacked plasma, platelet, and Proc. Natl. Acad. Sci. USA 103: 9649–9654. podocyte CFH. These mice developed spontaneous proliferative GN, 19. Bygrave, A. E., K. L. Rose, J. Cortes-Hernandez, J. Warren, R. J. Rigby, H. T. Cook, M. J. Walport, T. J. Vyse, and M. Botto. 2004. Spontaneous which could be accelerated in the CSS model. These disease features autoimmunity in 129 and C57BL/6 mice-implications for autoimmunity de- were unique relative to all other nonautoimmune strains studied in scribed in gene-targeted mice. PLoS Biol. 2: E243. the past (17, 18, 20–22, 24, 36, 37). It appeared that the presence of 20. Rose, K. L., D. Paixao-Cavalcante, J. Fish, A. P. Manderson, T. H. Malik, 2/2 A. E. Bygrave, T. Lin, S. H. Sacks, M. J. Walport, H. T. Cook, et al. 2008. human CR1 on Es altered IC processing in the CFH mouse, such Factor I is required for the development of membranoproliferative glomer- that there was increased IC deposition in glomeruli. Whether this ulonephritis in factor H-deficient mice. J. Clin. Invest. 118: 608–618. finding is physiologic or pathologic is not clear based on our data. 21. Alexander, J. J., O. G. B. Aneziokoro, A. Chang, B. K. Hack, A. Markaryan, A. Jacob, R. Luo, M. Thirman, M. Haas, and R. J. Quigg. 2006. Distinct and separable roles of Nonetheless, these ICs were not processed through the glomerular the in factor H-deficient bone marrow chimeric mice with im- capillary wall, wholly or in part because of the absence of CFH on mune complex disease. J. Am. Soc. Nephrol. 17: 1354–1361. The Journal of Immunology 9

22. Alexander, J. J., M. C. Pickering, M. Haas, I. Osawe, and R. J. Quigg. 2005. 29. Pemberton, M., G. Anderson, V. Ve˘tvicka, D. E. Justus, and G. D. Ross. 1993. Complement factor h limits immune complex deposition and prevents in- Microvascular effects of complement blockade with soluble recombinant CR1 flammation and scarring in glomeruli of mice with chronic serum sickness. on ischemia/reperfusion injury of skeletal muscle. J. Immunol. 150: 5104–5113. J. Am. Soc. Nephrol. 16: 52–57. 30. Kai, S., T. Fujita, I. Gigli, and V. Nussenzweig. 1980. Mouse C3b/C4b inacti- 23. Alexander, J. J., B. K. Hack, P. N. Cunningham, and R. J. Quigg. 2001. A protein vator: purification and properties. J. Immunol. 125: 2409–2415. with characteristics of factor H is present on rodent platelets and functions as the 31. Shin, M. L., M. C. Gelfand, R. B. Nagle, J. R. Carlo, I. Green, and M. M. Frank. 1977. Localization of receptors for activated complement on visceral immune adherence receptor. J. Biol. Chem. 276: 32129–32135. epithelial cells of the human renal glomerulus. J. Immunol. 118: 869–873. 24. Alexander, J. J., Y. Wang, A. Chang, A. Jacob, A. W. Minto, M. Karmegam, 32. Kazatchkine, M. D., D. T. Fearon, M. D. Appay, C. Mandet, and J. Bariety. 1982. M. Haas, and R. J. Quigg. 2007. Mouse podocyte complement factor H: the Immunohistochemical study of the human glomerular C3b receptor in normal functional analog to human complement receptor 1. J. Am. Soc. Nephrol. 18: kidney and in seventy-five cases of renal diseases: loss of C3b receptor antigen in 1157–1166. focal hyalinosis and in proliferative nephritis of systemic lupus erythematosus. 25. Repik, A., S. E. Pincus, I. Ghiran, A. Nicholson-Weller, D. R. Asher, J. Clin. Invest. 69: 900–912. A. M. Cerny, L. S. Casey, S. M. Jones, S. N. Jones, N. Mohamed, et al. 2005. 33. Moll, S., S. Miot, S. Sadallah, F. Gudat, M. J. Mihatsch, and J. A. Schifferli. A transgenic mouse model for studying the clearance of blood-borne 2001. No complement receptor 1 stumps on podocytes in human glomer- via human complement receptor 1 (CR1). Clin. Exp. Immunol. 140: 230–240. ulopathies. Kidney Int. 59: 160–168. 26. Panchuk-Voloshina, N., R. P. Haugland, J. Bishop-Stewart, M. K. Bhalgat, 34. Ren, G., M. Doshi, B. K. Hack, J. J. Alexander, and R. J. Quigg. 2003. Rat P. J. Millard, F. Mao, W. Y. Leung, and R. P. Haugland. 1999. Alexa dyes, glomerular epithelial cells produce and bear factor H on their surface that is up- a series of new fluorescent dyes that yield exceptionally bright, photostable regulated under complement attack. Kidney Int. 64: 914–922. conjugates. J. Histochem. Cytochem. 47: 1179–1188. 35. Akilesh, S., T. B. Huber, H. Wu, G. Wang, B. Hartleben, J. B. Kopp, J. H. Miner, 27. Rabellino, E. M., G. D. Ross, and M. J. Polley. 1978. Membrane receptors of D. C. Roopenian, E. R. Unanue, and A. S. Shaw. 2008. Podocytes use FcRn to clear IgG from the glomerular basement membrane. Proc. Natl. Acad. Sci. USA mouse leukocytes. I. Two types of complement receptors for different regions of 105: 967–972. C3. J. Immunol. 120: 879–885. 36. Bao, L., M. Haas, A. W. Minto, and R. J. Quigg. 2007. Decay-accelerating factor 28. Alsenz, J., D. Avila, H. P. Huemer, I. Esparza, J. D. Becherer, T. Kinoshita, but not CD59 limits experimental immune-complex glomerulonephritis. Lab. Y. Wang, S. Oppermann, and J. D. Lambris. 1992. Phylogeny of the third Invest. 87: 357–364. Downloaded from component of complement, C3: analysis of the conservation of human CR1, 37. Quigg, R. J., A. Lim, M. Haas, J. J. Alexander, C. He, and M. C. Carroll. 1998. CR2, H, and B binding sites, concanavalin A binding sites, and thiolester bond in Immune complex glomerulonephritis in C4- and C3-deficient mice. Kidney Int. the C3 from different species. Dev. Comp. Immunol. 16: 63–76. 53: 320–330. http://www.jimmunol.org/ by guest on September 23, 2021