Feb 17, 1996 - Female NZB/W F1 mice produce high titers of antinuclear antibodies and invariably succumb to severe glomerulonephritis by 12 months of age.
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)
confirmed a role for terminal complement activation in the progression of renal disease (5-7). We have been studying the contribution of activated terminal complement components to inflammatory disease processes, using monoclonal antibodies (mAbs) specific for CS (8, 9). Functionally, these mAbs inhibit the cleavage of CS, thus blocking the generation of the potent proinflammatory molecules C5a and CSb-9 (terminal complement complex), but not preventing the formation of C3b, which subserves critical immunoprotective functions of opsonization and immune complex clearance (10). In this study, we have directly examined the involvement of complement in the pathogenesis of the glomerulonephritis in CS sufficient NZB/W F1 mice, using a mAb specific for murine C5 (11). We show that continuOus treatment with an antimurine C5 mAb results in marked amelioration of the course of renal disease and in dramatic prolongation of survival. These results demonstrate an important role for activated terminal complement components in the immune complexmediated inflammatory disease of NZB/W F1 mice.
New Zealand black x New Zealand white ABSTRACT (NZB/W) F1 mice spontaneously develop an autoimmune syndrome with notable similarities to human systemic lupus erythematosus. Female NZB/W F1 mice produce high titers of antinuclear antibodies and invariably succumb to severe glomerulonephritis by 12 months of age. Although the development of the immune-complex nephritis is accompanied by abundant local and systemic complement activation, the role of proinflammatory complement components in disease progression has not been established. In this study we have examined the contribution of activated terminal complement proteins to the pathogenesis of the lupus-like autoimmune disease. Female NZB/W F1 mice were treated with a monoclonal antibody (mAb) specific for the C5 component of complement that blocks the cleavage of C5 and thus prevents the generation of the potent proinflammatory factors C5a and C5b-9. Continuous therapy with anti-C5 mAb for 6 months resulted in significant amelioration of the course of glomerulonephritis and in markedly increased survival. These findings demonstrate an important role for the terminal complement cascade in the progression of renal disease in NZB/W F1 mice, and suggest that mAb-mediated CS inhibition may be a useful approach to the therapy of immune-complex glomerulonephritis in humans.
The NZB/W F1 mouse develops a spontaneous autoimmune disease process with striking similarities to human systemic lupus erythematosus (SLE). In female NZB/W F1 mice, the production of IgG antinuclear antibodies, including antibodies to double-stranded DNA (dsDNA), is associated with the development of a severe immune complex-mediated glomerulonephritis that results in death from renal failure in virtually all animals by 12 months of age (1). Although various studies have explored the factors responsible for the onset of autoimmunity in these mice (2), little is known regarding the pathogenic mechanisms of the renal disease following immune complex deposition. For example, one hallmark of immune complex-initiated inflammation is the activation of the complement cascade through both the classical and alternative pathways (3). In NZB/W F1 mice, as in human SLE, the production of autoantibodies and consequent tissue deposition of immune complexes result in local and systemic complement activation sufficient in magnitude to cause a marked reduction in serum complement-dependent hemolytic activity (4). This observation clearly implicates the complement system in the pathogenesis of the immune complex nephritis. However, studies in NZB-derived mouse strains deficient in the C5 component of complement have not
MATERIALS AND METHODS Animals. Female NZB/W F1 mice (8- to 12-weeks old) were purchased from The Jackson Laboratory and were maintained under pathogen-free conditions. Antibodies and Treatment. The anti-mouse CS hybridoma BB5.1 as well as the control murine anfti-human C8 hybridoma 135.8 were described previously (9). Both hybridomas were grown as ascites in athymic mice and the antibodies were purified from ascites by protein A affinity chromatography followed by elution with ImmunoPure IgG elution buffer (Pierce) and dialysis against Tris-buffered saline. Concentrations of purified antibodies were.determined at O.D. 280 with a Beckman DU-640 spectrophotometer. At 18 weeks of age, mice were begun on biweekly treatments with 1 mg of either anti-CS or control mAb administered intraperitoneally. Beginning at 26 weeks of age, the frequency of treatments was increased to three times per week and from 32 weeks onward daily treatments were initiated. Mice were bled every 2 weeks for determination of serum hemolytic activity. Hemolytic assays were performed as described previously (9). The 100% value for complement-dependent serum hemolytic activity was determined using normal mouse serum (Sigma). Assays. Urine protein levels were determined three times per week by colorimetric analysis using dipsticks (Chemstrip 2GP, Boehringer Mannheim) and quantitated according to the following parameters: trace; 1+, 30 mg/dl; 2+, 100 mg/dl; 3 +, 500 mg/dl. Standard ELISA assays to measure the serum
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Abbreviations: SLE, systemic lupus erythematosus; H&E, hematoxylin and eosin; FcR, Fc receptor. TTo whom reprint requests should be addressed.
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levels, specificity, and isotype of anti-DNA antibodies were performed as previously described (12). Briefly, 96-well flat bottom ELISA plates (VWR Scientific) were coated with dsDNA (Sigma) or single-stranded DNA (Sigma) at 4°C overnight and were then blocked with bovine serum albumin (BSA) before incubation with various dilutions of serum samples obtained from NZB/W F1 mice. The plates were washed and then incubated with horseradish peroxidasecoupled goat anti-mouse Ig (Zymed). For isotype analysis of anti-dsDNA antibodies, horseradish peroxidase-coupled antibodies 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 incubation with substrate (O-phenylene-D-diamine for the horseradish peroxidase-coupled anti-mouse Ig antibody; and 2,2'-azino-di-[3-ethylbenzthiazoline-6-sulfonate] for the horseradish peroxidase-coupled antibodies in the Ig isotype determination kit), the plates were read at O.D. 490 nm and 405 nm, respectively. Baseline levels of anti-dsDNA and anti-single-stranded DNA antibodies were determined using sera from 5-week-old NZB/W F1 female mice before the onset of signs of autoimmune disease. The serum titers of anti-DNA antibodies in anti-C5 and control mAb-treated animals were measured at 18, 28, and 32 weeks. The results for each group were recorded as the fold increase in the number of O.D. units relative to the common baseline titer measured on the serum from the 5-week-old NZB/W F1 mice. Renal Histopathology. The kidneys from euthanized control mAb-treated, anti-C5 mAb-treated and young untreated mice were fixed in 10% buffered formalin. The tissue was then processed and embedded in paraffin with a VIP tissue processor (Miles). Tissue sections (5 gm) were stained with hematoxylin/eosin (H&E) or with periodic acid Schiff using standard methodology. Quantitative analyses of mesangial matrix deposition, glomerular crescent formation, and deposition of tubular casts, representative features of the renal histopathology in the NZB/W F1 model of lupus glomerulonephritis, were performed on tissue from control-mAb-treated mice, anti-C5 mAb-treated mice, and untreated 18-week-old NZB/W F1 mice (to determine the baseline values at the time treatment was begun). All samples were randomly chosen from equivalently sectioned specimens processed from mice and were read in a blinded fashion by two independent investigators. For analysis of mesangial matrix volume and crescent formation, 10 randomly selected H&E-stained glomeruli were analyzed per mouse. Images were captured using a JVC TK-1070U video system through a 40 x objective lens and were quantitated with IMAGE-PRO PLUS software (Media Cybernetics, Silver Spring, MD). To analyze mesangial matrix deposition, the perimeter of the glomerular mesangial matrix from each hematocylin/ eosin (H&E)-stained glomerulus was traced and the area determined by performing pixel counts. Mesangial cell areas were automatically excluded from the measurements using the color differentiation parameters of the count/size command of the software. Similarly coded glomerular images were also analyzed for the presence of crescent formation, and the percentage of glomeruli with crescents was calculated for each animal. Quantitative analysis of the number of tubular casts was performed by counting all the tubules in one coded 20X field obtained from a site on the renal cortex directly opposite the renal pelvis and determining the percentage of enlarged tubules filled with casts relative to the total number of tubules identified. Two independent counts by separate observers were performed on each kidney and the mean percentage of tubules filled with casts was calculated. Arthus Reactions. Reverse passive Arthus reactions were performed on anesthetized anti-CS mAb-treated and control mice. The mice were shaved and injected intravenously with 20 mg/kg of bovine serum albumin (Miles). They were then
Proc. Natl. Acad. Sci. USA 93 (1996)
injected intradermally with 100 gg of purified rabbit anti-BSA IgG or anti-OVA IgG (Cappel) or buffer alone in 50 ,lI. Intradermal injections of anti-BSA IgG were also performed in animals that had not received intravenous antigen as controls. After 4 hr, the areas of the macroscopic skin lesions were determined by multiplying the maximal transverse widths (mm) in two perpendiculkr directions. Skin biopsies were taken through the center of the lesions, fixed in 10% buffered formalin, H&E-stained, and then examined for edema, hemorrhage, and inflammatory cell infiltrates. RESULTS Inhibition of Complement in NZB/W F1 Mice. To examine the role of activated terminal complement components in the progression of autoimmune disease in NZB/W F1 female mice, 4-month-old animals were begun on biweekly treatments with either anti-C5 or an isotype matched control mAb. We have previously reported that the anti-C5 mAb blocks the generation of both CSa and CSb-9 (9). In vivo inhibition of complement by anti-C5 mAb was ascertained by serial measurement of complement-dependent serum hemolytic activity. The mAb treatments, continued until the mice were 40 weeks, were titered thereafter to maintain serum hemolytic activity at a level less than 10% of that of normal control mouse serum (Fig. 1A). At the time of initiation of therapy, all mice had low but measurable levels of circulating anti-dsDNA antibodies
(Fig. 1B). Anti-C5 mAb administration was able to sustain complement inhibition in vivo for the entire 6-month period of treatment, as measured by reduced serum hemolytic activity (Fig. 1A). In contrast to anti-C5 mAb-treated animals, the serum hemolytic activity of the control mAb-treated mice was normal at the outset of the study and then gradually declined to less than 10% of normal levels by 40 weeks, presumably secondary to systemic consumption of complement after widespread tissue deposition of immune complexes (Fig. 1A). The decline in hemolytic activity in the sera of the control animals correlated with elevated titers of anti-dsDNA antibodies measured over the same period of time (Fig. 1B). Both anti-C5 and control mAb-treated mice produced comparable amounts of anti-dsDNA antibodies, as measured relative to the same baseline serum sample (Fig. 1B). A much smaller, but detectable, increase in the level of anti-single-stranded DNA antibodies was also observed (Fig. 1B). Moreover, consistent with previous reports (13), the predominant isotype of the antidsDNA antibodies produced was IgG2a (Fig. 1C). The isotype pattern of anti-dsDNA antibody production was unaffected by anti-C5 mAb therapy (Fig. 1C). In addition, quantitative immunofluorescence demonstrated equivalent levels of immune complex and C3 deposition in the glomeruli of anti-C5 and control mAb-treated mice (data not shown). Amelioration of Immune Complex Glomerulonephritis and Prolongation of Survival by Anti-C5 Therapy. The influence of mAb-mediated C5 inhibition on the course of NZB/W F1 immune complex nephritis was examined both clinically and histopathologically. A marked delay in the onset of severe proteinuria, defined as equal to or greater than 500 mg/dl (.3+), was achieved in anti-C5 mAb-treated mice relative to control mAb-treated animals and untreated age-matched NZB/W F1 mice (Fig. 2A). Whereas all of the control mAbtreated and untreated mice had developed proteinuria accompanied by marked total body edema by 32 weeks, no anti-C5 mAb-treated animals developed proteinuria until 33 weeks, and a significant percentage of these mice maintained normal renal function without evidence of proteinuria throughout the treatment period (Fig. 2A). Coincident with ameliorating the clinical signs of severe immune complex nephritis, CS inhibition was associated with a dramatic prolongation of survival. Nearly 80% of anti-CS
Proc. Natl. Acad. Sci. USA 93 (1996)
Immunology: Wang et al.
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FIG. 2. (A) Anti-C5 mAb therapy delays onset of proteinuria in NZB/W F1 mice. Serial measurements of urine protein in untreated, control mAb-treated, and anti-C5 mAb-treated animals were performed. The data are presented as percentage of animals with .3+ urine protein. (B) Prolongation of survival following anti-C5 mAb treatment of NZB/W F1 autoimmune disease. Shown are the percentage of surviving animals in anti-C5 treated (n = 20), control mAb-treated (n = 27), and mAb-untreated (n = 8) groups of animals at different ages. (A and B) Untreated, (A); control mAb-treated, (0); anti-C5 mAb-treated, (0).
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FIG. 1. (A) In vivo inhibition of complement by anti-C5 mAb administration. Female NZB/W F1 mice were treated with anti-C5 or isotype matched control mAb beginning at 18 weeks as described. Sera from both treatment and control groups were tested for complement dependent hemolytic activity every 14 days until 40 weeks. (0), Control mAb-treated; (a), anti-C5 mAb-treated. (B) Progressive age-related elevation of serum anti-dsDNA and anti-single-stranded DNA antibody titers in NZB/W F1 mice. Antibody titers (O.D. units) were determined at 18, 28, and 32 weeks, and recorded as fold increase relative to a common baseline titer performed on serum from 5-weekold NZB/W F1 mice as described. Solid symbols, control mAb-treated; open symbols, anti-C5 mAb-treated; squares, anti-single-stranded DNA; circles, anti-dsDNA. (C) Titers of various isotypes of antidsDNA antibodies measured in sera of control mAb-treated and anti-C5 mAb-treated mice at three different time points. Assays were performed as described.
mAb-treated mice were still alive at 40 weeks, in contrast to
