Complement Factor H Limits Immune Complex Deposition and Prevents and Scarring in Glomeruli of Mice with Chronic

Jessy J. Alexander,* Matthew C. Pickering,† Mark Haas,‡ Iyabo Osawe,* Richard J. Quigg* *Section of Nephrology, The University of Chicago, Chicago, Illinois; †Rheumatology Section, Imperial College, Hammersmith Campus, London, United Kingdom; and the ‡Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland

Factor H is the major complement regulator in plasma. Abnormalities in factor H have been implicated in membranoprolif- erative in both humans and experimental animals. It has been shown that factor H on rodent platelets functions analogously to human erythrocyte 1 in its role to traffic immune complexes to the mononuclear system. C57BL/6 factor H-deficient mice (Cfh؊/؊) and wild-type (wt) controls were immunized daily for 5 wk with heterologous apoferritin to study the chronic serum sickness GN model. Immunizations were started in 6- to 8-wk-old mice, .which was before the development of spontaneous membranoproliferative glomerulonephritis in some Cfh؊/؊ animals Glomerular deposition of IgG immune complexes in glomeruli was qualitatively and quantitatively increased in Cfh؊/؊ mice compared with wt mice. Consistent with the increase in glomerular immune complexes and possibly because of alternative pathway complement activation, Cfh؊/؊ mice had increased glomerular C3 deposition. Wt mice developed no glomerular pathology. In contrast, Cfh؊/؊ mice developed diffuse proliferative GN with focal crescents and glomerulosclerosis. In .addition, there was significantly increased expression of collagen IV, fibronectin, and laminin mRNA in Cfh؊/؊ glomeruli These data show a role for platelet-associated factor H to process immune complexes and limit their accumulation in glomeruli. Once deposited in glomeruli, excessive complement activation can lead to glomerular inflammation and the rapid development of a scarring phenotype. J Am Soc Nephrol 16: 52–57, 2005. doi: 10.1681/ASN.2004090778

ctivation of the complement cascade leads to the pro- It is not surprising, given that factor H is a major fluid phase duction of a number of proteins that contribute to complement regulator, that abnormalities in this protein are A inflammation. This system is beneficial when contrib- associated with several renal diseases. The most consistent uting to host defense, but can be detrimental if activated on self associations are with membranoproliferative glomerulonephri- tissue. To prevent this, a number of naturally occurring com- tis (MPGN) and hemolytic uremic syndrome. Humans with plement regulatory proteins are present to restrict complement dysfunctional factor H molecules (10–12) and animals with activation throughout the cascades of the three pathways. The factor H deficiencies (13,14) develop renal disease with features regulators of complement activation family comprise a collec- of MPGN. These associations of abnormal factor H molecules tion of plasma- and cell-associated proteins that limit activation with glomerular disease support the commonly held belief that of C3 and C5 (1–3). Each family member has between 4 and 44 activation of the complement cascade contributes to immuno- tandemly arranged amino acid complement control protein logically mediated glomerular diseases (15,16). Additional cir- modules (4,5). An important member of this family is factor H, cumstantial evidence for this is the presence of complement ␤ which was originally isolated in 1965 as 1H globulin by Nils- activation products in glomeruli and in urine (17–19). The use son and Mu¨ller-Eberhard (6), and 10 yr later its function was of animal models has strengthened the case for a role of com- determined by both the Ruddy and Fearon laboratories (7,8). By plement activation in many glomerular diseases. virtue of its affinity for , factor H inhibits the formation and Serum sickness and the resultant GN have been studied in accelerates the decay of alternative pathway C3 convertases different animal species, using a variety of immunization pro- and serves as a cofactor for the factor I-mediated cleavage and tocols. Daily immunization with heterologous apoferritin leads inactivation of C3b (9). to crescentic GN in susceptible strains of mice (20), which can progress to glomerulosclerosis (21). A dependence on the pres-

Received September 18, 2004. Accepted October 24, 2004. ence of C5 was shown by Falk and Jennete in this model, implicating C5a and/or C5b-9 (22). Because the 129 and Published online ahead of print. Publication date available at www.jasn.org. C57BL/6 strains used for gene targeting are relatively resistant Address correspondence to: Dr. Jessy J. Alexander, Section of Nephrology, The to this model (see (23) and below), a modification was adopted University of Chicago, 5841 S. Maryland Avenue, MC5100, Chicago, IL 60637, Phone: 773-702-4796; Fax: 773-702-5818; E-mail: [email protected]. by Welch et al. in which lipopolysaccharide was administered uchicago.edu with apoferritin (24). In this model of progressive glomerular

Copyright © 2005 by the American Society of Nephrology ISSN: 1046-6673/1601-0052 J Am Soc Nephrol 16: 52–57, 2005 Factor H in Immune Complex Disease 53 and tubular inflammation, signaling of the C5a respectively; P ϭ 0.018 by t test). complexed in through its receptor on inflammatory cells and potentially on plasma immune complexes were also measured by virtue of renal tubular epithelium (25) led to tubulointerstitial disease their ability to bind solid-phase C1q (34). In contrast to free Ϫ Ϫ but did not affect glomerular disease (26). anti-apoferritin antibodies, Cfh / mice had no change in cir- In vitro studies have clearly shown that complement is re- culating immune complexes compared with baseline, and these quired for solubilization of large – immune were significantly less than that present in wt mice (0.26 Ϯ 0.03 Ϫ Ϫ complexes, presumably by disrupting lattice formation when and 0.68 Ϯ 0.11 U/ml in apoferritin-immunized Cfh / and wt C3 and C4 bind covalently to constituents of the complex (27). mice, respectively; P ϭ 0.024 by t test). These data illustrate the Complement also plays an important role in the in vivo traffick- complexities of the in the humoral immune ing of immune complexes. In humans, immune complexes response (35) and immune complex processing (27,28,34). bearing C4b and C3b bind to (CR1), Taken together, it appears that both groups of animals pro- which transports them to the cells of the mononuclear phago- duced anti-apoferritin IgG antibodies, but it was only in the cyte system in the liver and (28). In mice and other presence of functional factor H that immune complexes were subprimate species, the functional homologue of erythrocyte generated and retained in plasma. CR1, which we have identified as factor H (29), is present on We were then interested in whether factor H affected glo- platelets and not erythrocytes (30,31). In addition to affecting merular localization of IgG-containing immune complexes in the fate of systemic immune complexes, activation of C3 clearly this experimental serum sickness model. There was minimal Ϫ Ϫ can facilitate the processing of immune complexes directly in IgG in the glomeruli of Cfh / animals immunized with saline glomeruli (32). for 5 wk (Figure 1A), which was no different than wt animals (not shown). In glomeruli of wt mice immunized for 5 wk with Experimental Model apoferritin, there was a significant quantity of IgG in mesangia Ϫ Ϫ For these studies, C57BL/6 factor H-deficient (Cfh / ) mice along with extension to peripheral capillary loops (Figure 1B). Ϫ Ϫ generated and generously provided by Dr. Marina Botto (Ham- In Cfh / mice with serum sickness, there was a qualitative mersmith Hospital, London, UK) were used (14). These mice (Figure 1C) and quantitative difference (as scored in Figure 1D), have been backcrossed Ͼ10 generations onto the C57BL/6 in that factor H-deficient animals had more intense mesangial strain, including in our lab. As such, normal C57BL/6 mice staining as well as greater involvement in peripheral capillary (Jackson Laboratories, Bar Harbor, ME) were used as wild-type loops. (wt) strain controls in these studies. Immune complex GN was The reasons behind the increased quantities of IgG in glo- Ϫ Ϫ Ϫ Ϫ induced by immunizing 6- to 8-wk-old male Cfh / (n ϭ 10) or meruli of Cfh / mice are undoubtedly complex, as factor H wt (n ϭ 6) mice for 5 wk with a daily intraperitoneal dose of 4 has a number of potentially relevant functions. Factor H on mg horse spleen apoferritin (Sigma Chemical Co., St. Louis, mouse platelets appears to serve the function of the related CR1 MO), as we and others have described (20,22,23). Control protein on human erythrocytes (29); both have affinity for Ϫ Ϫ Cfh / (n ϭ 5) and wt (n ϭ 3) mice were treated identically, classical pathway-generated C3b on immune complexes (28,29). Ϫ Ϫ except they received the saline diluent alone. Although Cfh / Once bound to platelet factor H (or erythrocyte CR1), transfer mice on mixed 129 and C57BL/6 background are known to of immune complexes to the mononuclear phagocyte system is Ϫ Ϫ develop MPGN later in life (14), our studying Cfh / on a pure facilitated by factor H (or CR1) serving as cofactor for the factor C57BL/6 background at an early age made it unlikely that I-mediated cleavage of C3b to iC3b, which binds to CR3 on spontaneous GN would occur, which was confirmed in these (28,36). In human glomeruli, CR1 on podocytes also studies. There was no spontaneous mortality in the control serves an immune adherence function (37,38). As with the Ϫ Ϫ groups, while four of 10 Cfh / mice and one of six wt mice circulating immune adherence receptor, it is conceivable that actively immunized with apoferritin died over the 5-wk study the function of CR1 in human podocytes is served by factor H period. Although these results suggested that factor H could in rodents (39), which could include immune complex process- limit spontaneous mortality in this model (21), these differences ing within glomeruli. One other consideration is that due to Ϫ Ϫ were not statistically different. A total of 19 mice remained at unrestricted complement activation, Cfh / mice have ac- Ϫ Ϫ the end of 5 wk, of which 11 were Cfh / mice and eight were quired deficiency of C3 (14), which can profoundly affect im- wt mice. These animals were sacrificed and a comprehensive mune complex processing (23,40). However, in our past studies assessment of disease phenotype was performed, as we have applying this same serum sickness model in C3-deficient mice, described previously in this model (23) and in mice IgG deposition in glomeruli was considerably less in C3-defi- (33,34). cient mice compared with wt controls (23), making this mech- anism less likely. Whatever the mechanism(s), factor H defi- Results and Discussion ciency is associated with defective immune complex As expected, there were no anti-apoferritin antibodies in all processing, leading to a significant increase in immune complex groups at baseline and after 5 wk of saline administration. With retention in glomeruli. apoferritin immunization, all mice mounted a humoral im- In control wt mice given saline for 5 wk, there was no Ϫ Ϫ mune response. After 5 wk, Cfh / mice had greater quantities glomerular IgG or C3 (not shown), while in wt mice immunized of free anti-apoferritin IgG antibodies compared with wt ani- with apoferritin, staining for C3 was of similar distribution and Ϫ Ϫ mals (0.38 Ϯ 0.03 and 0.25 Ϯ 0.02 U/ml in Cfh / and wt mice, intensity (Figure 2B and scored in Figure 2D) as that for IgG 54 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 52–57, 2005

Ϫ Ϫ (Figure 1B), suggesting that glomerular IgG-containing im- way (41). Thus, it appears that in Cfh / mice with serum mune complexes activated the complement locally. In contrast, sickness, impaired systemic and local immune complex pro- Ϫ Ϫ although glomeruli of control Cfh / mice contained negligible cessing leads to a significant increase in glomerular IgG-con- quantities of IgG, there was significant glomerular C3 (Figure taining complexes, which are capable of activating the classical 2A). Even as these mice age and spontaneously develop complement pathway and contributing to unrestricted alterna- MPGN, C3 and C9 are present in subendothelial deposits while tive pathway activation. Ϫ Ϫ IgG is only present in mesangia (14). Thus, control Cfh / mice In spite of significant immune complex deposition and com- appear to have unrestricted alternative pathway complement plement activation, wt mice immunized with apoferritin had no activation in glomeruli rather than immune complex–directed histologic evidence for glomerular disease (Figure 3A). Thus, Ϫ Ϫ classical pathway activation. In Cfh / mice immunized with C57BL/6 mice are relatively resistant to the development of GN Ϫ Ϫ apoferritin, there was marked complement activation above in the setting of chronic serum sickness (23,24). Control Cfh / this baseline (Figure 2C). As with wt mice, IgG and C3 were mice receiving saline for 5 wk similarly had mild to no evident Ϫ Ϫ present together in Cfh / mice (in this case throughout the glomerular pathology (Figure 3B illustrates mesangial promi- glomerular capillary), supporting the idea that immune com- nence, the most severe abnormality in this group), which is plexes were responsible for this increased complement activa- consistent with the late development of MPGN in unmanipu- Ϫ Ϫ Ϫ Ϫ tion. C3 activation by IgG-bearing immune complexes should lated Cfh / mice (14). In contrast, all six Cfh / mice immu- occur through the classical pathway, which is not appreciably nized with apoferritin developed significant GN, characterized affected by factor H (but rather by C4-binding protein). How- by diffuse hypercellularity of the glomerular tufts with focal ever, there is growing evidence that such classical pathway crescents (Figure 3C, arrow), in addition to the presence of focal activation can contribute to activation of the alternative path- and segmental glomerular sclerosis/hyalinosis (Figure 3D). All

Figure 1. Factor H limits the accumulation of IgG-containing immune complexes in glomeruli of mice with chronic serum sickness. Ϫ Ϫ Shown is representative immunofluorescence staining for IgG in (A) control C57BL/6 factor H-deficient mice (Cfh / ) mice given Ϫ Ϫ saline for 5 wk, and (B) wild-type (wt) or (C) Cfh / mice immunized for 5 wk with apoferritin. Staining intensity scores compiled from all mice are also shown (D). *P Ͻ 0.002 versus all other groups by ANOVA followed by Fisher’s pairwise comparisons. Original magnifications, 200ϫ.

Figure 2. Functional factor H limits complement activation in glomeruli of normal mice and in those with chronic serum sickness. Ϫ Ϫ Shown is representative immunofluorescence staining for C3 in (A) control Cfh / mice given saline for 5 wk, and (B) wt or (C) Ϫ Ϫ Cfh / mice immunized for 5 wk with apoferritin. Staining intensity scores compiled from all mice are also shown (D). *P Ͻ 0.006 versus all other groups by ANOVA followed by Fisher’s pairwise comparisons (i.e. each group was different from another). Original magnification, 200ϫ. J Am Soc Nephrol 16: 52–57, 2005 Factor H in Immune Complex Disease 55

Figure 3. Factor H prevents the development of glomerular pathology in chronic serum sickness. (A) Wt mice immunized with Ϫ Ϫ apoferritin for 5 wk had normal glomerular and tubulointerstitial histology. (B) Control Cfh / mice administered saline for 5 wk Ϫ Ϫ had either normal glomeruli or mild mesangial changes. In contrast, Cfh / mice immunized for 5 wk with apoferritin developed GN, characterized by diffuse hypercellularity and focal crescent formation (C, arrow) as well as focal and segmental hyalinosis/ sclerosis (D, glomerulus at right). Scoring for the extent of glomerulonephritis (E), percent involvement of glomeruli with crescents (F), and sclerosis/hyalinosis (G) in all mice is also shown. *P Ͻ 0.001 and **P Ͻ 0.0025 versus all other groups by ANOVA followed by Fisher’s pairwise comparisons. Original magnification, 200ϫ (A), 400ϫ (B through D).

Ϫ Ϫ of these histopathological features were significantly different erritin-immunized wt and Cfh / mice, there was no difference than in the other groups (Figures 3, E through G). Consistent in albuminuria between the two groups. Hence, there is not a with the development of glomerular sclerosis in apoferritin- direct relationship between the presence of glomerular immune Ϫ Ϫ immunized Cfh / mice, there was an increase in mRNA for complexes, C3 deposition or glomerular sclerosis/hyalinosis, collagen IV, fibronectin, and laminin compared with the other and impaired glomerular permselectivity in this model. Al- groups as measured by quantitiative RT-PCR (Table 1). though blood urea nitrogen levels were elevated in apoferritin- Ϫ Ϫ Urinary albumin excretion in apoferritin-immunized wt mice immunized Cfh / mice, because of the variability of these was elevated compared with that of control animals, while values in this group of animals (range, 28 to 126 mg/dl), they blood urea nitrogen levels were normal (Table 2). These data were not statistically different from the other groups. Given the are consistent with our past findings in this model (23). In spite renal pathologic findings of significant GN and glomerular of the marked difference in renal pathologic findings in apof- sclerosis/hyalinosis (Ն4.3% glomerular involvement) in all six

Table 1. Expression of mRNA for matrix components in renal cortices of mice immunized with apoferritin or salinea

Immunogen Genotype n Laminin Fibronectin Collagen IV

Saline Wild-type 3 0.44 Ϯ 0.12 2.05 Ϯ 0.29 0.69 Ϯ 0.13 Ϫ Ϫ Cfh / 5 0.95 Ϯ 0.16 3.08 Ϯ 0.42 0.69 Ϯ 0.04 Apoferritin Wild-type 5 2.79 Ϯ 0.32b 4.65 Ϯ 0.57c 4.76 Ϯ 0.56c Ϫ Ϫ Cfh / 6 3.00 Ϯ 1.22b 6.38 Ϯ 0.54d 9.33 Ϯ 1.18e

Ϫ Ϫ aExpression of mRNA for the listed matrix components in renal cortices from wild-type and Cfh / mice after 5 wk of daily immunization with either saline or apoferritin was determined by quantitative (real-time) reverse-transcription–PCR and normalized to expression of 18S RNA from the same sample. Data are presented as mean Ϯ SEM and were analyzed by Ϫ Ϫ ANOVA followed by Fisher’s pairwise comparisons. Cfh / , C57BL/6 factor H-deficient. bP Ͻ 0.005 compared to saline groups. cP Ͻ 0.05 compared to saline groups. dP Ͻ 0.05 compared to all other groups. eP Ͻ 0.005 compared to all other groups. 56 Journal of the American Society of Nephrology J Am Soc Nephrol 16: 52–57, 2005

Table 2. Renal functional data in mice immunized with apoferritin or salinea

Immunogen Genotype n BUN (mg/dl) Albuminuria (␮g/mg creatinine)

Saline Wild-type 3 25.5 Ϯ 2.0 12.1 Ϯ 2.1 Ϫ Ϫ Cfh / 5 25.9 Ϯ 0.9 12.3 Ϯ 0.6 Apoferritin Wild-type 5 29.6 Ϯ 2.3 58.7 Ϯ 11.1b Ϫ Ϫ Cfh / 6 48.7 Ϯ 15.6 50.1 Ϯ 7.1b

Ϫ Ϫ aBUN and albuminuria levels were measured in wild-type and Cfh / mice after 5 wk of daily immunization with either saline or apoferritin. Data are mean Ϯ SEM. BUN, blood urea nitrogen. bAlbuminuria was increased in apoferritin-immunized mice compared to saline controls (P Ͻ 0.001) with no other statistically significant comparisons.

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