Amelioration of Lupus-Like Autoimmune Disease in NZB/W F1 Mice After
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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8563-8568, August 1996 Immunology Amelioration of lupus-like autoimmune disease in NZB/W F1 mice after treatment with a blocking monoclonal antibody specific for complement component C5 (immune complex/glomerulonephritis/inflammation) YI WANG*t, QILE Hu*, JOSEPH A. MADRIt, SCOrr A. ROLLINS*, AMY CHODERA*, AND Louis A. MATIS*t *Immunobiology Program, Alexion Pharmaceuticals, Inc., New Haven, CT 06511; and tDepartment of Pathology, Yale University School of Medicine, New Haven, CT 06510-8023 Communicated by David W. Talmage, Webb-Waring Institute for Biomedical Research, Denver, CO, May 6, 1996 (received for review February 17, 1996) ABSTRACT New Zealand black x New Zealand white confirmed a role for terminal complement activation in the (NZB/W) F1 mice spontaneously develop an autoimmune progression of renal disease (5-7). syndrome with notable similarities to human systemic lupus We have been studying the contribution of activated termi- erythematosus. Female NZB/W F1 mice produce high titers of nal complement components to inflammatory disease pro- antinuclear antibodies and invariably succumb to severe cesses, using monoclonal antibodies (mAbs) specific for CS (8, glomerulonephritis by 12 months of age. Although the devel- 9). Functionally, these mAbs inhibit the cleavage of CS, thus opment of the immune-complex nephritis is accompanied by blocking the generation of the potent proinflammatory mol- abundant local and systemic complement activation, the role ecules C5a and CSb-9 (terminal complement complex), but not of proinflammatory complement components in disease pro- preventing the formation of C3b, which subserves critical gression has not been established. In this study we have immunoprotective functions of opsonization and immune examined the contribution of activated terminal complement complex clearance (10). proteins to the pathogenesis of the lupus-like autoimmune In this study, we have directly examined the involvement of disease. Female NZB/W F1 mice were treated with a mono- complement in the pathogenesis of the glomerulonephritis in clonal antibody (mAb) specific for the C5 component of CS sufficient NZB/W F1 mice, using a mAb specific for murine complement that blocks the cleavage of C5 and thus prevents C5 (11). We show that continuOus treatment with an anti- the generation of the potent proinflammatory factors C5a and murine C5 mAb results in marked amelioration of the course C5b-9. Continuous therapy with anti-C5 mAb for 6 months of renal disease and in dramatic prolongation of survival. resulted in significant amelioration of the course of glomer- These results demonstrate an important role for activated ulonephritis and in markedly increased survival. These find- terminal complement components in the immune complex- ings demonstrate an important role for the terminal comple- mediated inflammatory disease of NZB/W F1 mice. ment cascade in the progression of renal disease in NZB/W F1 mice, and suggest that mAb-mediated CS inhibition may be a MATERIALS AND METHODS useful approach to the therapy of immune-complex glomeru- lonephritis in humans. Animals. Female NZB/W F1 mice (8- to 12-weeks old) were purchased from The Jackson Laboratory and were maintained under pathogen-free conditions. The NZB/W F1 mouse develops a spontaneous autoimmune Antibodies and Treatment. The anti-mouse CS hybridoma disease process with striking similarities to human systemic BB5.1 as well as the control murine anfti-human C8 hybridoma lupus erythematosus (SLE). In female NZB/W F1 mice, the 135.8 were described previously (9). Both hybridomas were production of IgG antinuclear antibodies, including antibodies grown as ascites in athymic mice and the antibodies were to double-stranded DNA (dsDNA), is associated with the purified from ascites by protein A affinity chromatography development of a severe immune complex-mediated glomer- followed by elution with ImmunoPure IgG elution buffer ulonephritis that results in death from renal failure in virtually (Pierce) and dialysis against Tris-buffered saline. Concentra- all animals by 12 months of age (1). tions of purified antibodies were.determined at O.D. 280 with Although various studies have explored the factors respon- a Beckman DU-640 spectrophotometer. At 18 weeks of age, sible for the onset of autoimmunity in these mice (2), little is mice were begun on biweekly treatments with 1 mg of either known regarding the pathogenic mechanisms of the renal anti-CS or control mAb administered intraperitoneally. Be- disease following immune complex deposition. For example, ginning at 26 weeks of age, the frequency of treatments was one hallmark of immune complex-initiated inflammation is the increased to three times per week and from 32 weeks onward activation of the complement cascade through both the clas- daily treatments were initiated. Mice were bled every 2 weeks sical and alternative pathways (3). In NZB/W F1 mice, as in for determination of serum hemolytic activity. Hemolytic human SLE, the production of autoantibodies and consequent assays were performed as described previously (9). The 100% tissue deposition of immune complexes result in local and value for complement-dependent serum hemolytic activity was systemic complement activation sufficient in magnitude to determined using normal mouse serum (Sigma). cause a marked reduction in serum complement-dependent Assays. Urine protein levels were determined three times hemolytic activity (4). This observation clearly implicates the per week by colorimetric analysis using dipsticks (Chemstrip complement system in the pathogenesis of the immune com- 2GP, Boehringer Mannheim) and quantitated according to the plex nephritis. However, studies in NZB-derived mouse strains following parameters: trace; 1+, 30 mg/dl; 2+, 100 mg/dl; 3+, deficient in the C5 component of complement have not 500 mg/dl. Standard ELISA assays to measure the serum The publication costs of this article were defrayed in part by page charge Abbreviations: SLE, systemic lupus erythematosus; H&E, hematox- payment. This article must therefore be hereby marked "advertisement" in ylin and eosin; FcR, Fc receptor. accordance with 18 U.S.C. §1734 solely to indicate this fact. TTo whom reprint requests should be addressed. 8563 Downloaded by guest on September 29, 2021 8564 Immunology: Wang et al. Proc. Natl. Acad. Sci. USA 93 (1996) levels, specificity, and isotype of anti-DNA antibodies were injected intradermally with 100 gg of purified rabbit anti-BSA performed as previously described (12). Briefly, 96-well flat IgG or anti-OVA IgG (Cappel) or buffer alone in 50 ,lI. bottom ELISA plates (VWR Scientific) were coated with Intradermal injections of anti-BSA IgG were also performed dsDNA (Sigma) or single-stranded DNA (Sigma) at 4°C in animals that had not received intravenous antigen as overnight and were then blocked with bovine serum albumin controls. After 4 hr, the areas of the macroscopic skin lesions (BSA) before incubation with various dilutions of serum were determined by multiplying the maximal transverse widths samples obtained from NZB/W F1 mice. The plates were (mm) in two perpendiculkr directions. Skin biopsies were washed and then incubated with horseradish peroxidase- taken through the center of the lesions, fixed in 10% buffered coupled goat anti-mouse Ig (Zymed). For isotype analysis of formalin, H&E-stained, and then examined for edema, hem- anti-dsDNA antibodies, horseradish peroxidase-coupled anti- orrhage, and inflammatory cell infiltrates. bodies to mouse IgM, IgA, IgGl, IgG2a, IgG2b, or IgG3 derived from a murine Ig isotype subtyping kit (Boehringer Mannheim) were added to the dsDNA coated wells. Following RESULTS incubation with substrate (O-phenylene-D-diamine for the Inhibition of Complement in NZB/W F1 Mice. To examine horseradish peroxidase-coupled anti-mouse Ig antibody; and the role of activated terminal complement components in the 2,2'-azino-di-[3-ethylbenzthiazoline-6-sulfonate] for the progression of autoimmune disease in NZB/W F1 female mice, horseradish peroxidase-coupled antibodies in the Ig isotype 4-month-old animals were begun on biweekly treatments with determination kit), the plates were read at O.D. 490 nm and either anti-C5 or an isotype matched control mAb. We have 405 nm, respectively. Baseline levels of anti-dsDNA and previously reported that the anti-C5 mAb blocks the genera- anti-single-stranded DNA antibodies were determined using tion of both CSa and CSb-9 (9). In vivo inhibition of comple- sera from 5-week-old NZB/W F1 female mice before the onset ment by anti-C5 mAb was ascertained by serial measurement of signs of autoimmune disease. The serum titers of anti-DNA of complement-dependent serum hemolytic activity. The mAb antibodies in anti-C5 and control mAb-treated animals were treatments, continued until the mice were 40 weeks, were measured at 18, 28, and 32 weeks. The results for each group titered thereafter to maintain serum hemolytic activity at a were recorded as the fold increase in the number of O.D. units level less than 10% of that of normal control mouse serum relative to the common baseline titer measured on the serum (Fig. 1A). At the time of initiation of therapy, all mice had low from the 5-week-old NZB/W F1 mice. but measurable levels of circulating anti-dsDNA antibodies Renal Histopathology. The kidneys from euthanized control (Fig. 1B). mAb-treated, anti-C5 mAb-treated and young untreated mice Anti-C5 mAb administration was able to sustain comple- were fixed